Macrocyclic Cysteine Protease Inhibitors and Compositions Thereof

ABSTRACT

The present invention provides a novel class of macrocyclic compounds, which are useful as cysteine protease inhibitors. Also provided are novel intermediates and methods of preparing the compounds. The invention also provides pharmaceutical compositions comprising the compounds. The compounds and compositions are useful in methods of treating or preventing one or more diseases associated with cysteine protease activity, particularly those associated with calpain activity.

TECHNICAL FIELD

This invention relates to compounds with enzyme inhibitory activity andto compositions that comprise one or more of these compounds. Inparticular, the invention relates to compounds that inhibit cysteineproteases, particularly calpains. These compounds have utility in thetreatment of a variety of diseases.

BACKGROUND ART

A protease is an enzyme that degrades proteins into smaller peptidefragments. Cysteine proteases incorporate a cysteine residue that isessential to the catalytic process.

Calpains are cysteine proteases that are activated by elevated levels ofintracellular calcium ions. Under normal circumstances, calcium ionsignalling of calpain leads to controlled proteolysis duringcytoskeletal remodelling, signal transduction and apoptosis in mammals.Uncontrolled or high levels of calcium ions in a cell can causeexcessive calpain activity, and lead to tissue damage.

There are two major isoforms of calpain, which require differentconcentrations of calcium ions for activity. They are μ-calpain (alsoknown as calpain I or calpain 1) and m-calpain (also known as calpain IIor calpain 2). The μ-calpain isoform has been identified as the majorisoform present during pathological conditions of the nervous systemsuch as Alzheimer's disease, motor neuron damage, muscular dystrophy andstroke.

The m-calpain isoform has been associated with the development ofcataracts. Cataracts are a condition whereby the lens of an eye becomesincreasingly clouded and eventually results in blindness. The cloudingis due to the precipitation of degraded lens proteins that results fromsustained activity of the calcium ion-activated calpain.

Several classes of calpain inhibitors are known. However, many of theknown calpain inhibitors have limited therapeutic potential because theyhave poor stability, cell permeability, solubility or selectivity, orbecause they have high cell toxicity.

Accordingly, it is an object of the present invention to go some way toavoiding the above disadvantages or to at least provide the public witha useful choice.

Other objects of the invention may become apparent from the followingdescription which is given by way of example only.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the prioritydate.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt, solvate, hydrate or prodrugderivative thereof:

wherein;

A is —C(═O)R₅ or —S(═O)₂R₆;

-   -   wherein R₅ is optionally substituted C₁-C₆ alkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted aryloxy, optionally substituted heteroaryloxy,        optionally substituted arylalkoxy or optionally substituted        heteroarylalkoxy; and    -   R₆ is optionally substituted C₁-C₆ alkyl, optionally substituted        aryl, optionally substituted heteroaryl, optionally substituted        aryloxy, optionally substituted heteroaryloxy, optionally        substituted arylalkoxy or optionally substituted        heteroarylalkoxy;        R₂ is a side chain of a natural or non-natural alpha-amino acid;        R₃ is —CH₂OH, —CH₂OR₇, —CH₂N₃, —CH₂NR₈R₉, —CH(OH)R₁₀, —CHO,        —CH(OH)C(═O)NR₁₁R₁₂, —C(═O)C(═O)NR₁₁R₁₂, or —C(═O)R₁₃;    -   wherein R₇ is C₁-C₆ alkyl, aryl or arylalkyl;    -   R₈ is hydrogen, C₁-C₆ alkyl, aryl or arylalkyl;    -   R₉ is hydrogen, C₁-C₆ alkyl, aryl or arylalkyl;    -   R₁₀ is C₁-C₆ alkyl, alkoxy, thioalkoxy, aryl, arylalkyl,        heteroaryl, heteroarylalkyl or cyano;    -   R₁₁ and R₁₂ are each independently selected from hydrogen, C₁-C₆        alkyl, hydroxyalkyl, aryl, arylalkyl, heterocyclyl,        heterocyclylalkyl, heteroaryl and heteroarylalkyl; or R₁₁ and    -   R₁₂ taken together with the nitrogen to which they are attached        form a heterocyclyl or heteroaryl; and    -   R₁₃ is C₁-C₆ alkyl, aryl, arylalkyl, heteroaryl or        heteroarylalkyl;        R₄ is —O—R₂₀— which is attached to the 1,4-phenylene ring        through the oxygen atom;    -   wherein R₂₀ is optionally substituted straight chain        —(C₃-C₆)-alkyl- or optionally substituted straight chain        —(C₃-C₆)-alkenyl-; wherein any one methylene group within the        straight chain —(C₃-C₆)-alkyl- or straight chain        —(C₃-C₆)-alkenyl-, except the methylene group adjacent to the        oxygen atom to which R₂₀ is attached, may be replaced by an        oxygen, nitrogen or sulfur heteroatom or a —S(═O)— or —S(═O)₂—        group; and wherein any two carbon atoms, or a carbon atom and a        nitrogen heteroatom, if present, of the straight chain        —(C₃-C₆)-alkyl- or straight chain —(C₃-C₆)-alkenyl- may be        linked to one another through a chain of 1 to 4 atoms to form a        fused ring selected from optionally substituted cycloalkyl,        optionally substituted cycloalkenyl, optionally substituted        heterocyclyl, optionally substituted aryl and optionally        substituted heteroaryl; and        R₆₀, R₆₁, R₆₂ and R₆₃ are each independently selected from        hydrogen, halogen, —NH₂, —NO₂, —OH, C₁-C₆ alkyl and C₁-C₆        alkoxy.

The present invention also provides a compound of Formula II or a salt,solvate or hydrate thereof:

wherein;

R₅₀ is —C(═O)O—R₄₀;

-   -   R₄₀ is alkyl, benzyl, allyl, dialkylphenyl or silyl;        A₁ is hydrogen, an amino protecting group or A; and        A, R₂, R₄, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I.

The present invention also provides a compound of Formula III or a salt,solvate or hydrate thereof:

wherein;A₁ is hydrogen or an amino protecting group; andR₂, R₃, R₄, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I.

The present invention also provides a compound of Formula IV or a salt,solvate or hydrate thereof:

wherein;R₂₁ is optionally substituted straight chain (C_(m))-alkyl; andR₂₂ is optionally substituted straight chain (C_(n))-alkyl;

-   -   wherein m=0-4 and n=0-4; provided that the sum m+n=1-4; and        wherein any one methylene group of R₂₁ and R₂₂, except that        adjacent to the oxygen atom to which R₂₂ is attached may be        replaced by an oxygen, nitrogen or sulfur heteroatom or a        —S(═O)— or —S(═O)₂— group; and wherein any two carbon atoms, or        a carbon atom and a nitrogen atom, if present, of R₂₁ may be        linked to one another through a chain of 1 to 4 atoms to form a        fused ring selected from optionally substituted cycloalkyl,        optionally substituted cycloalkenyl, optionally substituted        heterocyclyl, optionally substituted aryl and optionally        substituted heteroaryl, or any two carbon atoms, or a carbon        atom and a nitrogen atom, if present, of R₂₂ may be linked to        one another through a chain of 1 to 4 atoms to form a fused ring        selected from optionally substituted cycloalkyl, optionally        substituted cycloalkenyl, optionally substituted heterocyclyl,        optionally substituted aryl and optionally substituted        heteroaryl;        A₁ is hydrogen, an amino protecting group or A; and        A, R₂, R₆₀, R₆₁, R₆₂ and R₆₃ are, as defined for Formula I and        R₅₀ is as defined for Formula II.

The present invention also provides a compound of Formula V or a salt,solvate or hydrate thereof:

wherein R₂, R₆₀, R₆₁/R₆₂ and R₆₃ are as defined for Formula I and A₁ andR₂₂ are as defined for Formula IV.

The present invention also provides a compound of Formula VI or a salt,solvate or hydrate thereof:

wherein A₁ is hydrogen, an amino protecting group or A; andA, R₂, R₂₀, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I and R₅₀is as defined for Formula II.

The present invention also provides the compound(S)-2-[(S)-3-(4-allyloxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-propionylamino]-4-methyl-pentanoicacid methyl ester:

The present invention also provides the compound(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acidmethyl ester:

The present invention also provides the compound(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionicacid:

The present invention also provides the compound(S)-2-[(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-ethyl-pentanoicacid methyl ester:

The present invention also provides the compound(S)-6-hydroxy-2-{(S)-2-[(S)-3-(4-hydroxy-phenyl)-2-methyl-propionylamino]-4-methyl-pentanoylamino}-hexanoicacid methyl ester:

The present invention also provides a process for preparing a compoundof Formula I:

the process comprising the steps of:(a) cyclising a compound of Formula VI:

-   -   to provide a compound of Formula II:

and(b) converting the compound of Formula II into the compound of FormulaI.

In one embodiment, the process further comprises the steps of:

(a) providing a compound of Formula VII:

-   -   or an activated acid derivative thereof, wherein A₁ is hydrogen,        an amino protecting group or A; and    -   A, R₂, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I; and        (b) coupling the compound of Formula VII with a compound of        Formula VIII:

-   -   or an activated amino derivative thereof, wherein R₂₀ is as        defined for Formula I and R₅₀ is as defined for Formula II, to        provide the compound of Formula VI.

The present invention also provides a process for preparing a compoundof Formula I:

the process comprising the steps of:(a) cyclising a compound of Formula IV:

-   -   to provide a compound of Formula II:

and(b) converting the compound of Formula II into the compound of FormulaI.

In one embodiment, the process further comprises the steps of:

(a) providing a compound of Formula V:

-   -   or an activated acid derivative thereof; and        (b) coupling the compound of Formula V with a compound of        Formula IX:

-   -   or an activated amino derivative thereof, wherein R₂₁ is as        defined for Formula IV and R₅₀ is as defined for Formula II, to        provide the compound of Formula IV.

In another aspect, the present invention provides a compound of FormulaI for use as a medicament.

In another aspect, the present invention provides a compound of FormulaI for use as a cysteine protease inhibitor.

In another aspect, the present invention provides a method forinhibiting a cysteine protease in a mammal comprising the step ofadministering a compound of Formula I to the mammal.

In another aspect, the present invention provides a method for thetreatment or prophylaxis of a disease or disorder resulting fromexcessive cysteine protease activity in a mammal comprising the step ofadministering a compound of Formula Ito the mammal.

In another aspect, the present invention provides an in vitro method forinhibiting a cysteine protease comprising contacting the cysteineprotease with a compound of Formula I.

In another aspect, the present invention provides a method of inhibitinga cysteine protease in a cell comprising contacting the cell with aneffective amount of a compound of Formula I.

In another aspect, the present invention provides a use of a compound ofFormula I for the manufacture of a medicament for reducing the activityof a cysteine protease.

In another aspect, the present invention provides a use of a compound ofFormula I for the manufacture of a medicament for the treatment orprophylaxis of a disease or disorder resulting from excessive cysteineprotease activity.

In another aspect, the present invention provides a method for thetreatment or prophylaxis of cataracts in a mammal comprising the step ofadministering a compound of Formula I to the mammal.

In another aspect, the present invention provides a use of a compound ofFormula I for the manufacture of a medicament for the treatment orprophylaxis of cataracts.

In another aspect, the present invention provides a compositioncomprising a compound of Formula I.

This invention may also be said broadly to consist in the parts,elements and features referred to or indicated in the specification ofthe application, individually or collectively, and any or allcombinations of any two or more said parts, elements or features, andwhere specific integers are mentioned herein which have knownequivalents in the art to which this invention relates, such knownequivalents are deemed to be incorporated herein as if individually setforth.

The term “comprising”, or variations such as “comprises”, as used inthis specification and claims means “consisting at least in part of”.That is to say when interpreting statements in this specification andclaims which include that term, the features prefaced by that term ineach statement all need to be present but other features can also bepresent.

Although the present invention is broadly as defined above, thosepersons skilled in the art will appreciate that the invention is notlimited thereto and that the invention also includes embodiments ofwhich the following description gives examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the Figures inwhich:

FIG. 1 is a graph of the results of a fluorescence-based assay of theinhibition of m-calpain activity by(7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]-nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9);

FIG. 2 is a graph of the mean opacification scores against time fornormal ovine lenses in EMEM (control), ovine lenses treated with 5 mMcalcium in EMEM (Ca²⁺ only) and ovine lenses treated with 5 mM calciumin EMEM containing 1 μM(7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9) (Ca²⁺ with 9) during a 20 h experiment;

FIG. 3 is a photograph of an ovine lens that was pre-incubated with(7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9) before being treated with calcium chloride;

FIG. 4 is a photograph of an ovine lens that was treated with calciumchloride; and

FIG. 5 is a graph of the mean change in eye score over three months forthe treatment of three groups of lambs genetically predisposed tocataracts with ointments containing(7S,10S,13S)-7-hydroxymethyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]-nona-deca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (8),(7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9) or with a placebo ointment.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt, solvate, hydrate or prodrugderivative thereof:

wherein;

A is —C(═O)R₅ or —S(═O)₂R₆;

-   -   wherein R₅ is optionally substituted C₁-C₆ alkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted aryloxy, optionally substituted heteroaryloxy,        optionally substituted arylalkoxy or optionally substituted        heteroarylalkoxy; and    -   R₆ is optionally substituted C₁-C₆ alkyl, optionally substituted        aryl, optionally substituted heteroaryl, optionally substituted        aryloxy, optionally substituted heteroaryloxy, optionally        substituted arylalkoxy or optionally substituted        heteroarylalkoxy;        R₂ is a side chain of a natural or non-natural alpha-amino acid;        R₃ is —CH₂OH, —CH₂OR₇, —CH₂N₃, —CH₂NR₈R₉, —CH(OH)R₁₀, —CHO,        —CH(OH)C(═O)NR₁₁R₁₂, —C(═O)C(═O)NR₁₁R₁₂, or —C(═O)R₁₃;    -   wherein R₇ is C₁-C₆ alkyl, aryl or arylalkyl;    -   R₈ is hydrogen, C₁-C₆ alkyl, aryl or arylalkyl;    -   R₉ is hydrogen, C₁-C₆ alkyl, aryl or arylalkyl;    -   R₁₀ is C₁-C₆ alkyl, alkoxy, thioalkoxy, aryl, arylalkyl,        heteroaryl, heteroarylalkyl or cyano;    -   R₁₁ and R₁₂ are each independently selected from hydrogen, C₁-C₆        alkyl, hydroxyalkyl, aryl, arylalkyl, heterocyclyl,        heterocyclylalkyl, heteroaryl and heteroarylalkyl; or R₁₁ and    -   R₁₂ taken together with the nitrogen to which they are attached        form a heterocyclyl or heteroaryl; and    -   R₁₃ is C₁-C₆ alkyl, aryl, arylalkyl, heteroaryl or        heteroarylalkyl;        R₄ is —O—R₂₀— which is attached to the 1,4-phenylene ring        through the oxygen atom;    -   wherein R₂₀ is optionally substituted straight chain        —(C₃-C₆)-alkyl- or optionally substituted straight chain        —(C₃-C₆)-alkenyl-; wherein any one methylene group within the        straight chain —(C₃-C₆)-alkyl- or straight chain        —(C₃-C₆)-alkenyl-, except the methylene group adjacent to the        oxygen atom to which R₂₀ is attached, may be replaced by an        oxygen, nitrogen or sulfur heteroatom or a —S(═O)— or —S(═O)₂—        group; and wherein any two carbon atoms, or a carbon atom and a        nitrogen heteroatom, if present, of the straight chain        —(C₃-C₆)-alkyl- or straight chain —(C₃-C₆)-alkenyl- may be        linked to one another through a chain of 1 to 4 atoms to form a        fused ring selected from optionally substituted cycloalkyl,        optionally substituted cycloalkenyl, optionally substituted        heterocyclyl, optionally substituted aryl and optionally        substituted heteroaryl; and        R₆₀, R₆₁, R₆₂ and R₆₃ are each independently selected from        hydrogen, halogen, —NH₂, —NO₂, —OH, C₁-C₆ alkyl and C₁-C₆        alkoxy.

As used herein, the term “side chain of a natural or non-naturalalpha-amino acid” means the group R_(A) in a natural or non-naturalamino acid of formula NH₂—CH(R_(A))—COOH.

As used herein, the term “natural alpha-amino acid” includes the 20L-amino acids (or a residue thereof) which commonly comprise mostpolypeptides in living systems, that is: alanine (Ala); arginine (Arg);asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln);glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ileu);leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe);proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine(Tyr); and valine (Val). The term also includes rarer amino acids foundin fibrous proteins (for example, 4-hydroxyproline, 5-hydroxylysine,N-methyllysine, 3-methylhistidine, desmosine and isodesmosine), andnaturally occurring amino acids not found in proteins (for example,gamma-aminobutyric acid, homocysteine, homoserine, citrulline,ornithine, canavanine, djenkolic acid and beta-cyanoalanine).

Natural alpha-amino acids which contain functional substituents, forexample amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl orindolyl groups in their characteristic side chains include arginine,lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine,threonine, tyrosine and cysteine. When R₁ and/or R₂ in the compounds ofthe invention is a side chain that includes a functional substituent,such as a side chain of one of those natural alpha-amino acids, thefunctional substituent may optionally be protected. Suitable protectinggroups are known to those skilled in the art.

As used herein, the term “non-natural alpha-amino acid” includes anyalpha-amino acid (or residue thereof) other than the natural amino acidslisted above. Non-natural amino acids include the D-isomers of thenatural L-amino acids. Non-natural amino acids also include, but are notlimited to: D-phenylalanine; norleucine; hydroxyproline;alpha-carboxyglutamic acid; and pyroglutamic acid.

The prefixes “D-” or “L-” indicate an alpha-amino acid of D- orL-configuration respectively. A “D.L-” prefix indicates a racemicmixture of amino acids of the two configurations. Where no prefix isincluded, this means that the amino acid can be of either the D- or theL-configuration, except in the Examples where residues are ofL-configuration unless otherwise stated.

As used herein, the term “pharmaceutically acceptable salt” is intendedto include acid addition salts of any basic moiety that may be presentin a compound of Formula I, and base addition salts of any acidic moietythat may be present in a compound of Formula I. Such salts are generallyprepared by reacting the compound with a suitable organic or inorganicacid or base. Examples of pharmaceutically acceptable salts of basicmoieties include: sulfates; methanesulfonates; acetates; hydrochlorides;hydrobromides; phosphates; toluenesulfonates; citrates; maleates;succinates; tartrates; lactates; and fumarates. Examples ofpharmaceutically acceptable salts of acidic moieties include: ammoniumsalts; alkali metal salts such as sodium salts and potassium salts; andalkaline earth metal salts such as calcium salts and magnesium salts.Other pharmaceutically acceptable salts will be apparent to thoseskilled in the art.

As used herein, the term “prodrug derivative” is intended to includefunctional derivatives of the compounds of Formula I, thepharmacological action of which results from conversion to a compound ofFormula I by metabolic processes within the body. Therefore, a prodrugderivative is any covalently bonded carrier that releases a compound ofFormula I in vivo when the prodrug derivative is administered to amammal. Prodrug derivatives are generally prepared by modifyingfunctional groups in such a way that the modification is cleaved in vivoto yield the parent compound. Conventional procedures for the selectionand preparation of suitable prodrug derivatives are known to thosepersons skilled in the art and are discussed in, for example, T. Higuchiand V. Stella, Pro-drugs as Novel Delivery Systems, volume 14 of theA.C.S. Symposium Series, 1987, and E. B. Roche (ed.), BioreversibleCarriers in Drug Design, Pergamon Press, New York, 1987.

The compounds of Formula I may form hydrates, or solvates withpharmaceutically acceptable solvents. The present invention contemplatessuch hydrates and solvates as well as the corresponding unsolvatedforms.

As used herein, the term “optionally substituted” is intended to meanthat one or more hydrogen atoms in the group indicated is replaced withone or more independently selected suitable substituents, provided thatthe normal valency of each atom to which the optional substituent/s areattached is not exceeded, and that the substitution results in a stablecompound.

Unless a moiety of a compound is defined as being unsubstituted, thatmoiety may be optionally substituted. In a preferred embodiment, theoptional substituents are independently selected from the groupconsisting of alkyl, alkoxyalkyl, aminoalkyl, haloalkyl, hydroxyalkyl,alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, arylalkyl, arylalkoxy,aryloxy, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkoxy,heteroarylalkyl, heteroaryloxy, carboxy, oxo, acyl, amido, nitro, cyano,hydroxyl and halo; —O(C═O)—R^(x), —C(═O)O—R^(x), —C(═O)—R^(x),NH—C(═O)—R^(x), —S(═O)—R^(x) and S(═O)₂—R^(x), wherein each R^(x) isindependently selected from alkyl, aryl, heterocyclyl and heteroaryl;—NR^(y)R^(z), -alkyl-NR^(y)R^(z), —C(═O)—NR^(y)R^(z), —S(═O)—NR^(y)R^(z)and —S(═O)₂—NR^(y)R^(z), wherein each R^(y) and R^(z) is independentlyselected from hydrogen, alkyl, aryl, heterocyclyl and heteroaryl.

The general chemical terms used in the formulae herein have their usualmeanings. For example:

As used herein, the term “alkyl” is intended to include straight chain,branched chain or cyclic saturated hydrocarbon groups. In oneembodiment, preferred alkyl groups comprise 1 to 6 carbon atoms. Inanother preferred embodiment, the alkyl group is methyl, ethyl,n-propyl, iso-propyl, cyclopropyl, n-butyl, iso-butyl, tert-butyl orcyclobutyl.

As used herein, the term “alkenyl” is intended to include straightchain, branched chain or cyclic mono-unsaturated hydrocarbon groups

As used herein, the term “alkoxy” is intended to include the groupsalkyl-O— where alkyl is as defined above.

As used herein, the term “aryl” is intended to include aromatic radicalsincluding, but not limited to: phenyl; naphthyl; indanyl; biphenyl; andthe like. In one embodiment, preferred aryl groups comprise 4 to 10carbon atoms.

As used herein, the term “aryloxy” is intended to include the groupsaryl-O— where aryl is as defined above.

As used herein, the term “arylalkoxy” is intended to include the groupsaryl-alkyl-O— where alkyl and aryl are as defined above.

As used herein, the term “arylalkyl” is intended to include the groupsaryl-alkyl- where alkyl and aryl are as defined above.

As used herein, the term “heteroaryl” is intended to includeheteroaromatic radicals including, but not limited to: pyrimidinyl;pyridyl; pyrrolyl; furyl; oxazolyl; thiophenyl; and the like.

As used herein, the term “heteroaryloxy” is intended to include thegroups heteroaryl-O— where heteroaryl is as defined above.

As used herein, the term “heteroarylalkoxy” is intended to include thegroups heteroaryl-alkyl-O— where alkyl and heteroaryl are as definedabove.

As used herein, the term “heteroarylalkyl” is intended to include thegroups heteroaryl-alkyl- where alkyl and heteroaryl are as definedabove.

As used herein, the term “heterocyclyl” is intended to includenon-aromatic saturated heterocyclic radicals including, but not limitedto: piperidinyl; pyrrolidinyl; piperazinyl; 1,4-dioxanyl;tetrahydrofuranyl; tetrahydrothiophenyl; and the like.

As used herein, the term “heterocyclylalkyl” is intended to include thegroups heterocyclyl-alkyl- where alkyl and heterocyclyl are as definedabove.

As used herein, the term “thioalkoxy” is intended to include the groupsalkyl-S— where alkyl is as defined above.

In one embodiment wherein R₅ or R₆ comprises a substituted aryl orheteroaryl group, the substituent/s on the aryl or heteroaryl group areindependently selected from —NH₂, —OH, alkyl, alkoxy, preferablymethoxy, and halogen.

In one embodiment wherein R₁₀ comprises a substituted aryl or heteroarylgroup, the substituent/s on the aryl or heteroaryl group areindependently selected from alkyl, alkoxy and halogen.

In a preferred embodiment wherein A is —C(═O)R₅, R₅ is optionallysubstituted arylalkoxy. More preferably, R₅ is benzyloxy.

In a preferred embodiment wherein A is —C(═O)R₅, R₅ is optionallysubstituted aryl or heteroaryl. More preferably, R₅ is 2-pyrrolyl.

In a preferred embodiment wherein A is —S(═O)₂R₆, R₆ is optionallysubstituted C₁-C₆ alkyl. In a further preferred embodiment, R₆ ismethyl.

In a preferred embodiment wherein A is —S(═O)₂R₆, R₆ is optionallysubstituted aryl. In a further preferred embodiment, R₆ is substitutedphenyl. More preferably, R₆ is halophenyl, more preferably,4-fluorophenyl.

In a preferred embodiment, R₂ is a hydrophobic side chain of a naturalor non-natural alpha-amino acid.

In a preferred embodiment, R₂ is a side chain of a natural alpha-aminoacid.

In a preferred embodiment, R₂ is alkyl.

In a preferred embodiment, R₂ is a side chain of L-leucine or L-valine.

In one embodiment wherein R₃ is —CH(OH)C(═O)NR₁₁R₁₂ or—C(═O)C(═O)NR₁₁R₁₂, R₁₁ and R₁₂ taken together with the nitrogen atom towhich they are attached form a heterocyclyl.

In a preferred embodiment wherein R₃ is —CH(OH)C(═O)NR₁₁R₁₂ or—C(═O)C(═O)NR₁₁R₁₂, one of R₁₁ and R₁₂ is hydrogen.

In a preferred embodiment wherein R₃ is —CH(OH)C(═O)NR₁₁R₁₂ or—C(═O)C(═O)NR₁₁R₁₂, R₁₁ is C₁-C₆ alkyl and R₁₂ is hydrogen. Morepreferably, R₁₁ is cyclopropyl, ethyl or methyl.

In a preferred embodiment wherein R₃ is —CH₂NR₈R₉, one of R₈ or R₉ iscyclopropyl.

In a preferred embodiment, R₃ is —CH₂OH or —CHO.

In a preferred embodiment, R₂₀ is unsubstituted straight chainC₃-C₆-alkyl.

In a preferred embodiment, R₂₀ is unsubstituted straight chainC₃-C₆-alkenyl.

In a preferred embodiment, R₂₀ is unsubstituted straight chainC₃-C₆-alkyl wherein one of the methylene groups has been replaced by —O—or —S—.

In one embodiment, wherein R₂₀ includes a fused ring, the fused ringincludes 3 to 8 ring members, more preferably 3 to 6 ring members.

In a particularly preferred embodiment, R₄ is —O-propyl-, —O-butyl-,—O-pentyl-, —O—(CH₂)₄OCH₂— or —O—(CH₂)₄SCH₂—.

In a preferred embodiment wherein R₃ is —CH(OH)R₁₀, R₁₀ is alkoxy orthioalkoxy. More preferably, R₁₀ is alkoxy. Still more preferably, R₁₀is C₁₆-C₁₈-alkoxy.

In another preferred embodiment wherein R₃ is —CH(OH)R₁₀, R₁₀ is cyano.

In a preferred embodiment, R₆₀, R₆₁, R₆₂ and R₆₃ are each hydrogen.

A preferred group of compounds of Formula I has the followingstereochemistry:

Another preferred group of compounds of Formula I has the followingstructural formula:

A particularly preferred group of compounds of Formula I consists of:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.

A further particularly preferred group of compounds of Formula Iconsists of:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.

Another particularly preferred group of compounds of Formula I consistsof:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.

The present invention also provides a compound of Formula II or a salt,solvate or hydrate thereof:

wherein;

R₅₀ is —C(═O)O—R₄₀;

-   -   R₄₀ is alkyl, benzyl, allyl, dialkylphenyl or silyl;        A₁ is hydrogen, an amino protecting group or A; and        A, R₂, R₄, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I.

The compounds of Formula II are useful as intermediates in thepreparation of compounds of Formula I.

In a preferred embodiment, R₄₀ is C₁-C₆-alkyl. More preferably, R₄₀ ismethyl or tert-butyl. More preferably, R₄₀ is methyl.

In a preferred embodiment, A₁ is tert-butoxycarbonyl, benzyloxycarbonylor —S(═O)₂-(4-fluorophenyl).

The present invention also provides a compound of Formula III or a salt,solvate or hydrate thereof:

wherein;A₁ is hydrogen or an amino protecting group; andR₂, R₃, R₄, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I.

The compounds of Formula III are useful as intermediates in thepreparation of compounds of Formula I.

In a preferred embodiment, A₁ is tert-butoxycarbonyl orbenzyloxycarbonyl.

The present invention also provides a compound of Formula IV or a salt,solvate or hydrate thereof:

wherein;R₂₁ is optionally substituted straight chain (C_(m))-alkyl; andR₂₂ is optionally substituted straight chain (C_(n))-alkyl;

-   -   wherein m=0-4 and n=0-4; provided that the sum m+n=1-4; and        wherein any one methylene group of R₂₁ and R₂₂, except that        adjacent to the oxygen atom to which R₂₂ is attached may be        replaced by an oxygen, nitrogen or sulfur heteroatom or a        —S(═O)— or —S(═O)₂— group; and wherein any two carbon atoms, or        a carbon atom and a nitrogen atom, if present, of R₂₁ may be        linked to one another through a chain of 1 to 4 atoms to form a        fused ring selected from optionally substituted cycloalkyl,        optionally substituted cycloalkenyl, optionally substituted        heterocyclyl, optionally substituted aryl and optionally        substituted heteroaryl, or any two carbon atoms, or a carbon        atom and a nitrogen atom, if present, of R₂₂ may be linked to        one another through a chain of 1 to 4 atoms to form a fused ring        selected from optionally substituted cycloalkyl, optionally        substituted cycloalkenyl, optionally substituted heterocyclyl,        optionally substituted aryl and optionally substituted        heteroaryl;        A₁ is hydrogen, an amino protecting group or A; and        A, R₂, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I and        R₅₀ is as defined for Formula II.

In a preferred embodiment, A₁ is tert-butoxycarbonyl, benzyloxycarbonylor —S(═O)₂-(4-fluorophenyl).

The compounds of Formula IV are useful as intermediates in thepreparation of compounds of Formula I, Formula II and Formula III.

The present invention also provides a compound of Formula V or a salt,solvate or hydrate thereof:

wherein R₂, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I and A₁and R₂₂ are as defined for Formula IV.

The compounds of Formula V are useful as intermediates in thepreparation of compounds of Formula I, Formula II, Formula III andFormula IV.

The present invention also provides a compound of Formula VI or a salt,solvate or hydrate thereof:

wherein A₁ is hydrogen, an amino protecting group or A; andA, R₂, R₂₀, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I and R₅₀is as defined for Formula II.

In a preferred embodiment, A₁ is benzyloxycarbonyl.

The compounds of Formula VI are useful as intermediates in thepreparation of compounds of Formula I, Formula II and Formula III.

The group R₅₀ in the compounds of Formulae II, IV and VI is a carboxylprotecting group. The present invention also contemplates compounds ofFormulae II′, IV′ and VI′ in which the group R₅₀ is an alternativecarboxyl protecting group such as; an oxazole (described in, forexample, H. L. Wehrmeister, J. Org. Chem. 1961, 26, 3821); a dioxanone(described in, for example, K. Ishihara et al., Synlett. 1996, 839); anortho ester (described in, for example, E. J. Corey and N. Raju,Tetrahedron Lett. 1983, 24, 5571); or a Braun ortho ester (described in,for example, D. Waldmuller, M. Braun and A. Steigel, Synlett. 1991,160); and wherein all of the other variables are as defined for thecorresponding compounds of Formulae II, IV and VI.

The present invention also provides the compound(S)-2-[(S)-3-(4-allyloxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-propionylamino]-4-methyl-pentanoicacid methyl ester:

The present invention also provides the compound(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acidmethyl ester:

The present invention also provides the compound(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionicacid:

The present invention also provides the compound(S)-2-[(S)-3-(4-but-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-ethyl-pentanoicacid methyl ester:

The present invention also provides the compound(S)-6-hydroxy-2-{(S)-2-[(S)-3-(4-hydroxy-phenyl)-2-methyl-propionylamino]-4-methyl-pentanoylamino}-hexanoicacid methyl ester:

The compounds of the invention may have asymmetric carbon atoms.Therefore, stereoisomers (both enantiomers and diastereomers) of suchcompounds can exist. The present invention contemplates the purestereoisomers and any mixture of the isomers. For example, a pureenantiomer of a compound of the invention can be isolated from a mixtureof enantiomers of the compound using conventional optical resolutiontechniques. Enol forms and tautomers are also contemplated.

Compounds of the invention, including compounds of Formula I, may beprepared by, for example, the process shown in Scheme 1. Thesubstituents R₆₀, R₆₁, R₆₂ and R₆₃ are omitted in the interests ofclarity.

As shown in Scheme 1, an orthogonally protected tri-peptide diene may besynthesised using the required C-terminus protected or side chainallylated amino acids. P represents an amino protecting group. StandardHATU mediated peptide coupling and base hydrolysis procedures may beemployed. The unsaturated macrocycle may then be formed from the dieneby, for example, thermal or microwave assisted ring closing metathesis.The unsaturated macrocycle can then be hydrogenated and the N-terminusprotecting group may be cleaved to yield the saturated macrocyclicamine. The address region moiety (A) may be introduced using eitherstandard sulfonamide or amide bond formation conditions. The methylester is then converted into the desired functional group (R₃).

Alternatively, compounds of the invention, including compounds ofFormula I, may be prepared by, for example, the process shown in Scheme2. The substituents R₆₀, R₆₁, R₆₂ and R₆₃ are omitted in the interestsof clarity.

As shown in Scheme 2, the required tri-peptide diene with the desiredaddress moiety (A) may be synthesised using the required C-terminusprotected or side chain allylated amino acids. P represents an aminoprotecting group. Standard HATU mediated peptide coupling and basehydrolysis procedures may be employed. The A group may be introducedusing either standard peptide coupling or sulfonyl chloride couplingprocedures. The unsaturated macrocycle may be formed from the diene by,for example, thermal or microwave assisted ring closing metathesis.

Hydrogenation and then conversion of the C-terminus protecting groupinto the desired functional group (R₃) may be used to yield the compoundof Formula I.

Alternatives to microwave assisted ring closing metathesis are known tothose skilled in the art. These include, for example: nucleophilicsubstitution reactions, such as the Mitsunobu reaction (described in,for example, A. Arasappan et al., J. Org. Chem. 2002, 67, 3923 and A.Arasappan et al., Bioorganic & Medicinal Chemistry Letters 2006, 16,3960); and palladium cross coupling reactions (described in, forexample, R. Bates, Organic Synthesis using Transition Metals, SheffieldAcademic Press Ltd., Sheffield, 2000).

Reagents and Conditions: (i) NaI, K₂CO₃/DMF; (ii) PPh₃,1,1′-(azodicarbonyl)dipiperidine; (iii) (CH₃SO)₂O,diisopropylethylamine, DCM; (iv) (CF₃SO)₂O, diisopropylethylamine, DCM;(v) Zn, Pd(Ph₃)₄, NaOH; (vi) Pd—C, H₂.

Scheme 3 illustrates several of the alternative ring closing reactions.The substituents R₆₀, R₆₁, R₆₂ and R₆₃ are omitted in the interests ofclarity. The nucleophilic substitution reactions involve a leavinggroup. Exemplary leaving groups are: halogen (i); triphenylphosphineoxide (Mitsunobu reaction) (ii); mesylate (iii); and triflate (iv). Thepalladium cross-coupling reaction is illustrated by the Negishi reaction(v).

The cyclisation of a dihydroxy intermediate of Formula VI, asexemplified in (ii), (iii) and (iv) in Scheme 3, is a particularlypreferred alternative to thermal or microwave assisted ring closingmetathesis.

The present invention also provides a process for preparing a compoundof Formula I:

the process comprising the steps of:(a) cyclising a compound of Formula VI:

-   -   to provide a compound of Formula II:

and(b) converting the compound of Formula II into the compound of FormulaI.

In one embodiment, the process further comprises the steps of:

(a) providing a compound of Formula VII:

-   -   or an activated acid derivative thereof, wherein A₁ is hydrogen,        an amino protecting group or A; and    -   A, R₂, R₆₀, R₆₁, R₆₂ and R₆₃ are as defined for Formula I; and        (b) coupling the compound of Formula VII with a compound of        Formula VIII:

-   -   or an activated amino derivative thereof, wherein R₂₀ is as        defined for Formula I and R₅₀ is as defined for Formula II, to        provide the compound of Formula VI.

The present invention also provides a process for preparing a compoundof Formula I:

the process comprising the steps of:(a) cyclising a compound of Formula IV:

-   -   to provide a compound of Formula II:

and(b) converting the compound of Formula II into the compound of FormulaI.

In one embodiment, the process further comprises the steps of:

(a) providing a compound of Formula V:

-   -   or an activated acid derivative thereof; and        (b) coupling the compound of Formula V with a compound of        Formula IX:

-   -   or an activated amino derivative thereof, wherein R₂₁ is as        defined for Formula IV and R₅₀ is as defined for Formula II, to        provide the compound of Formula IV.

The peptide coupling reactions are generally conducted indimethylformamide (DMF) in the presence of a coupling agent, such asO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), and a suitable base, such as diisopropylethyl amine (DIPEA). A catalytic auxiliary nucleophile, such as1-hydroxybenzotriazole (HOBt), may also be used. Alternatively, the acidchloride, acid fluoride or mixed acid anhydride of the carboxylic acidmay be utilised. Other coupling reagents may also be utilised including,but not limited to: N,N′-dicyclohexylcarbodiimide (DCC);N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC);(7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyAoP); bromotripyrrolidino-phosphonium hexafluorophosphate (PyBroP);and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU).

Compounds of the invention in which R₂₀ includes a carbon-carbon doublebond may be transformed into other compounds of the invention usingmethods known to those skilled in the art. For example, the double bondmay be halogenated (see, for example, P. B. D. de la Mare, ElectrophilicHalogenation, Cambridge University Press, Cambridge, 1976), epoxidised(see, for example, R. C. Larock, Comprehensive organic transformations,VCH, New York, 1989, p 456 and the references cited therein) ordihydroxylated (see, for example, R. C. Larock, Comprehensive organictransformations, VCH, New York, 1989, p 494 and the references citedtherein). The double bond may also be subjected to a Diels-Aldercycloaddition reaction to give compounds wherein R₂₀ includes a cyclicmoiety (see, for example, A. Wasserman, Diels-Alder Reactions, Elsevier,New York, 1965).

Compounds of the invention wherein R₃ is —CH(OH)R₁₀ and R₁₀ is alkoxy orthioalkoxy may be prepared by reacting the corresponding aldehyde withthe appropriate alcohol or thiol.

Compounds of the invention may be prepared according to the generalmethodology described above and in the Examples. A person skilled in theart will be able, without undue experimentation and with regard to thatskill and this disclosure, to select appropriate reagents and conditionsto modify these methodologies to produce compounds of the invention.

Those persons skilled in the art will appreciate that other syntheticroutes may be used to synthesize the compounds of the invention. Inaddition, those persons skilled in the art will appreciate that, in thecourse of preparing the compounds of the invention, the functionalgroups of intermediate compounds may need to be protected by protectinggroups. Functional groups which it may be desirable to protect include,but are not limited to: hydroxyl; amino; and carboxylic acid groups.Protecting groups may be added and removed in accordance with techniquesthat are well known to those persons skilled in the art. The use ofprotecting groups is described in, for example, J. W. F. McOmie (ed.),Protective Groups in Organic Chemistry, Plenum Press, London, 1973 andT. W. Greene and P. G. M. Wutz, Protective Groups in Organic Synthesis,2^(nd) edition, Wiley, New York, 1991.

As described in the Examples, compounds of Formula I have beendetermined to have enzyme inhibitory activity in tests which arepredictive of such activity in mammals, including humans. Suchproperties render the compounds of the invention suitable for use, aloneor together with other active agents, in a number of therapeuticapplications, including those that involve cysteine protease inhibition.

Molecular modelling of compounds of Formula I has demonstrated that theyare able to form the beta-strand typical peptide secondary structuralmotif. Without wishing to be bound by theory, it is believed that abeta-strand structure of a compound is required for molecularrecognition by, and inhibition of, cysteine proteases.

In particular, compounds of Formula I have been found to inhibitcalpains. There is experimental evidence to demonstrate the involvementof excessive calpain activity in a variety of pathologies (K. K. W.Wang, and P-W. Yuen, Trends Pharmacol. Sci. 1994, 15, 412; D. Brömme,Drug News Perspect. 1999, 12, 73). Such pathologies include:inflammatory and immunological diseases, for example rheumatoidarthritis, pancreatitis, multiple sclerosis and inflammations of thegastro-intestinal system including ulcerative or non-ulcerative colitisand Crohn's disease; cardiovascular and cerebrovascular diseases, forexample arterial hypertension, septic shock, cardiac or cerebralinfarctions of ischemic or hemorrhagic origin, ischemia, and disorderslinked to platelet aggregation; disorders of the central or peripheralnervous system, for example neurodegenerative diseases includingcerebral or spinal cord trauma, sub-arachnoid haemorrhage, epilepsy,ageing, senile dementia including Alzheimer's disease and Huntington'schorea, Parkinson's disease and peripheral neuropathies; osteoporosis;muscular dystrophies; cachexia; proliferative diseases, for exampleatherosclerosis or recurrence of stenosis; loss of hearing; oculardisorders, for example optic neuropathies, including ischemic opticneuropathies and diabetic neuropathy, glaucoma, macular degeneration andretinal damage, including detachment, tears or holes, presbyopia andcataracts; organ transplant; auto-immune and viral diseases, for examplelupus, AIDS, parasitic and viral infections, diabetes and itscomplications and multiple sclerosis; and cancer.

Given the role of calpains in these pathologies, the compounds ofFormula I can produce beneficial or favourable effects in theirtreatment.

Accordingly, in another aspect, the invention provides a compound ofFormula I for use as a medicament.

More particularly, the invention provides a compound of Formula I foruse as a cysteine protease inhibitor. In a preferred embodiment, thecysteine protease is a calpain.

In another aspect, the present invention provides a method forinhibiting a cysteine protease in a mammal comprising the step ofadministering a compound of Formula I to the mammal.

The term “mammal” as used herein refers to a human or non-human mammal.Examples of non-human mammals include livestock animals such as sheep,cows, pigs, goats, rabbits and deer; and companion animals such as cats,dogs, rodents and horses.

In another aspect, the present invention provides a method for thetreatment or prophylaxis of a disease or disorder resulting fromexcessive cysteine protease activity in a mammal comprising the step ofadministering a compound of Formula Ito the mammal.

The invention further provides an in vitro method for inhibiting acysteine protease comprising contacting the cysteine protease with acompound of the invention.

The invention also provides a method of inhibiting a cysteine proteasein a cell comprising contacting the cell with an effective amount of acompound of Formula I.

In another aspect, the present invention provides a use of a compound ofFormula I for the manufacture of a medicament for reducing the activityof a cysteine protease.

In another aspect, the present invention provides a use of a compound ofFormula I for the manufacture of a medicament for the treatment orprophylaxis of a disease or disorder resulting from excessive cysteineprotease activity.

In preferred embodiments of the method and use aspects of the presentinvention, the cysteine protease is a calpain.

In particular embodiments, the disease or disorder results fromexcessive calpain activity and is selected from the group consisting of:disorders of the central or peripheral nervous system; musculardystrophies; cachexia; loss of hearing; and ocular disorders.

In a particularly preferred embodiment, the disease or disorderresulting from excessive calpain activity is cataracts.

Therefore, in another aspect, the present invention provides a methodfor the treatment or prophylaxis of cataracts in a mammal comprising thestep of administering a compound of Formula Ito the mammal.

The present invention also provides a use of a compound of Formula I forthe manufacture of a medicament for the treatment or prophylaxis ofcataracts.

In another aspect, the invention provides a composition comprising acompound of Formula I. In a preferred embodiment, the composition is apharmaceutical composition and further comprises a pharmaceuticallyacceptable carrier, diluent or excipient.

Pharmaceutically acceptable carriers, diluents and excipients arenon-toxic to recipients at the dosages and concentrations employed. Eachcarrier, diluent and excipient must also be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation.

The compound of Formula I, or the composition comprising same, may beadministered to a mammal by different routes. The most suitable routemay depend upon, for example, the condition and disease of the mammal.Preferred administration routes are oral, parenteral and topical,including intraocular.

The compositions of the present invention may be formulated foradministration in unit dosage forms, such as tablets, capsules, pills,powders, granules, suppositories, sterile parenteral solutions orsuspensions, sterile non-parenteral solutions or suspensions, oralsolutions or suspensions, topical solutions or suspensions, andintraocular solutions or suspensions and the like, that comprise acompound of the invention as an active ingredient.

Solid or fluid unit dosage forms can be prepared for oraladministration.

Powders may be prepared by comminuting the active ingredient to asuitably fine size and mixing with a similarly comminuted diluent orexcipient. Suitable diluents and excipients are known to those personsskilled in the art.

Capsules may be produced by preparing a powder mixture as describedabove and filling into formed gelatine sheaths. Soft gelatine capsulesmay be prepared by encapsulating a slurry of the active ingredient withan acceptable vegetable oil, light liquid petrolatum or other inert oilor triglyceride.

Tablets may be made by preparing a powder mixture, granulating orslugging, adding a lubricant and pressing into tablets. The powdermixture is prepared by mixing the active ingredient, suitablycomminuted, with a diluent or base. Suitable diluents and bases areknown to those persons skilled in the art. The powder mixture can begranulated by wetting with a binder and forcing through a screen. As analternative to granulating, the powder mixture can be slugged, i.e. runthrough a tablet machine and the resulting imperfectly formed tabletsbroken into pieces (slugs). The slugs can be lubricated to preventsticking to the tablet-forming dies. The lubricated mixture can then becompressed into tablets.

In one embodiment, the tablet is provided with a protective coating.

Fluid unit dosage forms for oral administration, such as syrups, elixirsand suspensions, wherein a specific volume of composition contains apredetermined amount of active ingredient for administration, can beprepared. Water-soluble active ingredients can be dissolved in anaqueous vehicle together with other ingredients to form a syrup. Anelixir is prepared by using a hydro-alcoholic vehicle. Suspensions canbe prepared from insoluble forms in a suitable vehicle with the aid of asuspending agent.

Fluid unit dosage forms are prepared for parenteral administrationutilising an active ingredient and a sterile vehicle. The activeingredient can be either suspended or dissolved in the vehicle,depending on the form and concentration used. In preparing solutions,the water-soluble active ingredient can be dissolved in a suitablesolvent for injection and filter sterilised before filling into asuitable vial or ampoule and sealing. Adjuvants can also be dissolved inthe vehicle. Parenteral suspensions are prepared in substantially thesame manner.

In addition to oral and parenteral administration, the rectal andvaginal routes may be utilised. An active ingredient can be administeredby means of a suppository. A vehicle which has a melting point at aboutbody temperature or one that is readily soluble can be utilised.

Fluid unit dosage forms for intranasal instillation are preparedutilising an active ingredient and a suitable pharmaceutical vehicle.Alternatively, a dry powder can be utilised for insufflation.

The active ingredients, together with a gaseous or liquefied propellantand suitable adjuvants as may be necessary or desirable, can be packagedinto a pressurized aerosol container for use as an aerosol.

Suitable dosage forms for intraocular administration include, but arenot limited to: eye drops; and ophthalmic emulsions and ointments. Inaddition to a compound of the invention, the topical dosage forms maycomprise a variety of other components, for example: solvents;stabilisers; emulsifiers; suspending agents; surfactants; preservatives;buffers; isotonising agents; pH control agents; and ointment bases.

Examples of the techniques and protocols mentioned above can be found inA. R. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18^(th)edition, Mack Publishing Company, Easton 1990.

The compounds of Formula I and compositions of the invention may be usedin combination therapies with one or more other active agents. The oneor more other active agents may form part of the same composition, or beformulated as one or more separate compositions for administration atthe same time or a different time.

Administration of the compound of Formula I or composition of theinvention is preferably in a therapeutically effective amount, thisbeing an amount sufficient to show the desired benefit to the mammal,including preventing or alleviating the symptoms of any disease ordisorder being prevented or treated. The particular dosage of activeingredient to be administered will depend upon the specific disease tobe treated, and various characteristics of the mammal, including age,gender, health and weight. In addition, therapeutic factors such as thesite of delivery, the method of administration, any concurrenttreatment, the frequency of treatment and therapeutic ratio, may also berelevant. Determining the appropriate dosage is within the ability ofthose persons skilled in the art.

It is expected that a useful unit dosage will comprise between about 0.1to about 1000 mg, preferably 1 to 200 mg, of a compound of Formula I.

When the compound of Formula I is formulated for intraocularadministration, for example as an eye drop solution, it is expected thata useful concentration of a compound of the invention will compriseabout 0.001 to about 2.0% (w/v), preferably 0.01 to 1.0% (w/v).Approximately 20 to 50 μL of such a solution may be instilled into theeye at regular intervals throughout the day.

In a preferred embodiment, the compound of Formula I is formulated intoan ointment for intraocular administration. In a particularly preferredembodiment, the ointment has the following composition (w/w):

1% compound of Formula I25% cetyl stearyl alcohol35% wool fat39% paraffinum subl.

In a preferred embodiment, the compound of Formula I is formulated intoan emulsion for intraocular administration. In a particularly preferredembodiment, the emulsion has the following composition (w/w):

0.7% compound of Formula I20% cetyl stearyl alcohol25% wool fat25% paraffinum subl.1% sodium lauryl sulfate0.1% sodium benzoate28.3% water

The following non-limiting examples are provided to illustrate thepresent invention and in no way limit the scope thereof.

EXAMPLES

Compounds within the scope of the invention were prepared by thefollowing synthetic procedures.

Abbreviations

-   1,1,2-TCE 1,1,2-trichloroethane-   BOC tert-butoxycarbonyl-   BODIPY    4,4-difluoro-5,7-dimethyl-4-bora-3a,4-diaza-s-indacene-3-propionic    acid-   CBZ benzyloxycarbonyl-   1,2-DCE 1,2-dichloroethane-   DCM dichloromethane-   DIBAL-H diisobutylaluminium hydride-   DIPEA diisopropyl ethyl amine-   DMF dimethylformamide-   DMSO dimethylsulfoxide-   EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide-   EMEM Eagle's minimum essential medium-   ES electrospray mass spectrometry-   Et₂O diethyl ether-   EtOAc ethyl acetate-   FTIR Fourier transform infrared spectroscopy-   GSGC Grubbs second generation    catalyst—benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinyidene]dichloro(tricyclohexyl-phosphine)ruthenium-   h hour(s)-   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HOBt hydroxybenzotriazole-   HRMS high resolution mass spectrometry-   LCMS liquid chromatography mass spectrometry-   MeOH methanol-   min(s) minute(s)-   m.p. melting point-   NMR nuclear magnetic resonance spectroscopy-   Ph phenyl-   rt room temperature-   SO₃.Pyr sulfur trioxide.pyridine complex-   TFA trifluoroacetic acid-   THF tetrahydrofuran

Synthesis

Reagents and Conditions: (i) HATU, DIPEA, Leu-OMe, DMF, (80%); (ii)NaOH, THF, H₂O, MeOH, (97%); (iii) HATU, DIPEA, (s)-allyl-Gly-OMe, DMF,(97%); (iv) 3×10 mol % GSGC, 10 mol % chloro-dicyclohexyl borane,1,1,2-TCE, microwave, (91%); (v) H₂, 20 mol % Pd/C, MeOH, EtOAc, (98%);(vi) 4M HCl, 1,4-dioxane, (100%); (vii) benzyl chloroformate, DIPEA,DMF, (43%); (viii) LiAlH₄, THF, (83%); (ix) SO₃.Pyr, DIPEA, DMSO, DCM,(42%).

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-methyl-pentanoicacid methyl ester (1)

N—BOC—O-allyl-tyrosine (obtained from NeoMPS, San Diego, Calif. 92126,USA) (10.0 g, 32.5 mmol), HATU (12.4 g, 35.8 mmol) and leucine methylester hydrochloride (5.90 g, 65.0 mmol) were dissolved in anhydrous DMF(50 mL). DIPEA (22.7 mL, 130 mmol) was added and the reaction mixturewas stirred at rt for 18 h before being partitioned between EtOAc and 1Mhydrochloric acid. The organic phase was then washed sequentially with1M hydrochloric acid and brine before being dried (MgSO₄), filtered andconcentrated in vacuo. Purification was achieved using flashchromatography, eluting with a gradient of 1/5 EtOAc/(50/70) petroleumether to 100% EtOAc to yield a white solid, 8.45 g, 58%. R_(f)=0.33 (1/3EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.08 (2H, d J=8.5 Hz, Ar—H), 6.79 (2H, d J=8.5Hz, Ar—H), 6.43 (1H, d J=7.6 Hz, NH Leu), 6.04 (1H, tdd J=5.3 Hz, J=5.3Hz, J=10.5, J=17.1 Hz, CH₂CHCH₂), 5.26-5.42 (2H, m, CH₂CHCH₂), 5.09 (1H,d J=6.6 Hz, NH Tyr); 4.54-4.57 (1H, m, CH Leu), 4.47 (2H, d J=5.3 Hz,CH₂CHCH₂), 4.30-4.36 (1H, m, CHCH₂Ph), 3.66 (3H, s, CO₂CH₃), 2.88-3.02(2H, m, CHCH₂Ph), 1.54-1.62 (2H, m, CHCH₂CH(CH₃)₂), 1.43-1.51 (1H, m,CHCH₂CH(CH₃)₂) 1.38 (9H, s, (CH₃)₃), 0.91 (3H, d J=6.6 Hz, CHCH₂(CH₃)₂),0.89 (3H, d J=6.6 Hz, CHCH₂(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 174.1, 172.8, 171.4, 157.5, 155.5, 133.2,130.4, 130.3, 128.7, 117.4, 114.7, 68.7, 54.3, 52.1, 50.7, 41.3, 37.3,28.2, 24.6, 22.7, 21.8.

HRMS (ES) 449.2662 (MH⁺). C₂₄H₃₆N₂O₆ requires 449.2651.

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-methyl-pentanoicacid (2)

Methyl ester 1 (8.45 g, 18.8 mmol) was dissolved in THF (35 mL) andsodium hydroxide (1.13 g, 28.2 mmol) pre-dissolved in water (10 mL) wasadded. A further 15 mL of THF and 20 mL of methanol were added to obtaina homogenous solution. The reaction mixture was stirred at rt for 18 hbefore being concentrated in vacuo. The residue was partitioned betweenEtOAc and 1M hydrochloric acid. The aqueous phase was extracted twicemore with EtOAc and the combined organic extracts were washed withbrine, dried (MgSO₄), filtered and concentrated in vacuo to yield awhite foam, 7.25 g, 89%.

¹H-NMR (500 MHz in CDCl₃) 7.10 (2H, d J=8.5 Hz, Ar—H), 6.82 (2H, d J=8.5Hz, Ar—H), 6.58 (1H, d J=7.9 Hz, NH Leu), 6.03 (1H, tdd J=5.3 Hz, J=5.3Hz, J=10.5 Hz, J=17.3 Hz, CH₂CHCH₂), 5.25-5.42 (2H, m, CH₂CHCH₂), 5.22(1H, bs, NH Tyr), 4.54-4.58 (1H, m, CHCH₂CH(CH₃)₂), 4.48 (2H, d J=5.2Hz, CH₂CHCH₂), 4.34-4.40 (1H, m, CHCH₂Ph), 2.96-3.02 (2H, m, CHCH₂Ph),1.58-1.70 (2H, m, CHCHCH₂(CH₃)₂), 1.50-1.56 (1H, m, CHCHCH₂(CH₃)₂), 1.39(9H, s, C(CH₃)₃), 0.92 (3H, d J=6.2 Hz, CHCHCH₂(CH₃)₂), 0.91 (3H, dJ=6.2 Hz, CHCHCH₂(CH₃)₂).

LRMS (ES) 435.2 (MH⁺). C₂₃H₃₄N₂O₆ requires 435.2.

(S)-2-{(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-methyl-pentanoylamino}-pent-4-enoicacid methyl ester (3)

Carboxylic acid 2 (2.86 g, 6.58 mmol), HATU (2.75 g, 7.24 mmol) and(S)-allyl-glycine methyl ester hydrochloride (1.20 g, 7.24 mmol) weredissolved in DMF (30 mL). DIPEA was added (4.60 mL, 26.4 mmol) and thereaction mixture was stirred at rt for 18 h before being partitionedbetween EtOAc and 1M hydrochloric acid. The organic phase was washedsequentially with 1M hydrochloric acid and brine before being dried(MgSO₄), filtered and concentrated in vacuo. Purification was achievedusing flash chromatography, eluting with a gradient of 1/3 EtOAc/(50/70)petroleum ether to 2/1 EtOAc/(50/70) petroleum ether to yield a whitesolid, 1.40 g, 39%. R_(f)=0.70 (1/1 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.09 (2H, d J=8.5 Hz, Ar—H), 6.82 (2H, d J=8.5Hz, Ar—H), 6.65 (1H, d J=7.6 Hz, NH Gly), 6.47 (1H, d J=8.1 Hz, NH Leu),6.04 (1H, tdd J=5.3 Hz, J=5.3 Hz, J=10.5 Hz, J=17.0 Hz, OCH₂CHCH₂),5.62-5.71 (1H, m, CHCH₂CHCH₂), 5.08-5.44 (4H, m, OCH₂CHCH₂ andCHCH₂CHCH₂), 4.99 (1H, d J=7.3 Hz, NH Tyr), 4.57-4.63 (1H, m,CHCH₂CH(CH₃)₂), 4.49 (2H, d J=5.2 Hz, OCH₂CHCH₂), 4.41-4.47 (1H, m,CHCH₂CHCH₂), 4.25-4.34 (1H, m, CHCH₂Ph), 3.73 (3H, s, CO₂CH₃); 2.96-3.05(2H, m, CHCH₂Ph), 2.45-2.61 (2H, m, CHCH₂CHCH₂), 1.52-1.68 (2H, m,CHCHCH₂(CH₃)₂) 1.43-1.50 (1H, m, CHCHCH₂(CH₃)₂) 1.39 (9H, s, C(CH₃)₃),0.90 (3H, d J=6.4 Hz, CHCHCH₂(CH₃)₂), 0.89 (3H, d J=6.4 Hz,CHCHCH₂(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 171.8, 171.5, 171.3, 157.5, 155.4, 133.2,132.2, 132.0, 130.3, 130.1, 128.5, 119.2, 119.0, 117.6, 114.8, 80.2,68.7, 55.6, 52.3, 51.7, 40.9, 40.7, 37.0, 36.2, 28.2, 24.4, 22.8, 22.0.

HRMS (ES) 546.3180 (MH⁺). C₂₉H₄₃N₃O₇ requires 546.3179.

(E/Z)-(7S,10S,13S)-13-tert-Butoxycarbonylamino-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),4,15(19),16-tetraene-7-carboxylicacid methyl ester (4)

Diene 3 (1.40 g, 2.57 mmol) was dissolved in 1,1,2-trichloroethane (150mL) under an atmosphere of argon. Chloro-dicyclohexyl borane (0.257 mL,0.257 mmol) and GSGC (0.218 g, 0.257 mmol) were added. The reactionmixture was heated at reflux in the microwave (1200 W) for 20 mins. Twofurther additions of GSGC (0.218 g, 0.257 mmol) were added and aftereach the reaction mixture was subjected to a further 20 mins heating inthe microwave before being cooled and concentrated in vacuo.Purification was achieved using flash chromatography eluting with agradient of 1/2 EtOAc/(50/70) petroleum ether to 100% EtOAc to yield abrown solid, 0.379 g, 29%. The product was obtained as a mixture of E/Zisomers. R_(f)=0.70 (2/1 EtOAc/(50/70) petroleum ether).

¹H-NMR for major isomer from mixture (500 MHz in CDCl₃) 7.01 (2H, dJ=5.4 Hz, Ar—H), 6.78 (2H, d J=5.4 Hz, Ar—H), 5.87 (1H, d J=8.6 Hz, NHGly), 5.81 (1H, d J=7.1 Hz, NH Leu), 5.40-5.58 (2H, m, OCH₂CHCHCH₂ andOCH₂CHCHCH₂), 5.34 (1H, d J=8.6 Hz, NH Tyr), 4.73 (1H, ddd J=3.2 Hz,J=8.6 Hz, J=9.2 Hz, CHCO₂CH₃), 4.55-4.68 (2H, m, OCH₂CHCHCH₂), 4.09-4.20(2H, m, CHCH₂Ph and CHCH₂CH(CH₃)₂), 3.74 (3H, s, CO₂CH₃), 3.08 (1H, ddJ=4.9 Hz, J=12.6 Hz, CHCH₂Ph), 2.65-2.74 (1H, m, CHCH₂Ph), 2.26-2.34(2H, m, OCH₂CHCHCH₂), 1.82-1.90 (1H, m, CHCH₂CH(CH₃)₂), 1.52-1.58 (2H,m, CHCH₂CH(CH₃)₂), 1.44 (9H, s, (CH₃)₃), 0.84-0.88 (6H, m,CHCH₂CH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 3.77 (3H, s, CO₂CH₃),2.48-2.52 (1H, m, CHCH₂Ph), 1.48 (9H, s, (CH₃)₃), 0.89-0.94 (6H, m,CH₂CH(CH₃)₂).

HRMS (ES) 518.2869 (MH⁺). C₂₇H₃₉N₃O₇ requires 518.2866.

(7S,10S,13S)-13-tert-Butoxycarbonylamino-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo-[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (5)

Olefin 4 (0.379 g, 0.732 mmol) was dissolved in 40 mL of EtOAc. 10%palladium on carbon catalyst was added (0.0948 g, 25%) and the reactionmixture was subjected to hydrogenation at rt and atmospheric pressurefor 18 h before being filtered through celite and concentrated in vacuoto yield a brown solid, 0.341 g, 90%.

¹H-NMR (500 MHz in CDCl₃) 7.05 (2H, d J=7.8 Hz, Ar—H), 6.79 (2H, d J=7.8Hz, Ar—H), 6.08 (1H, d J=6.1 Hz NH Gly), 5.74 (1H, d J=8.2 Hz, NH Leu),5.22 (1H, d J=8.8 Hz, NH Tyr), 4.38-4.42 (1H, m, CHCH₂Ph), 4.31-4.36(1H, m, CHCO₂CH₃), 4.12-4.28 (2H, m, OCH₂CH₂CH₂CH₂), 3.96-4.06 (1H, m,CHCH₂CH(CH₃)₂), 3.73 (3H, s, CO₂CH₃), 3.11 (1H, dd J=5.3 Hz, J=12.2 Hz,CHCH₂Ph), 2.65 (1H, dd J=12.2 Hz, J=12.2 Hz CHCH₂Ph), 1.68-1.74 (2H, m,OCH₂CH₂CH₂CH₂), 1.49-1.63 (3H, m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 1.45(9H, s, C(CH₃)₃), 1.20-1.40 (2H, m, OCH₂CH₂CH₂CH₂), 0.82-0.85 (6H, m,CHCH₂CH(CH₃)₂).

HRMS (ES) 520.3031 (MH⁺). C₂₇H₄₁N₃O₇ requires 520.3023.

(7S,10S,13S)-13-Amino-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1-(18),15(19),16-triene-7-carboxylicacid methyl ester hydrogen chloride salt (6)

Methyl ester 5 (0.340 g, 0.654 mmol) was dissolved in 4M HCl in1,4-dioxane (20 mL). The solution was stirred at rt for 16 h beforebeing concentrated in vacuo to yield a brown solid, 0.232 g, 78%.

¹H-NMR (500 MHz in (CD₃)₂SO) 8.46 (2H, bs, NH₂), 8.32 (1H, d J=8.7 Hz NHLeu), 7.95 (1H, d J=7.8 Hz, NH Gly), 6.80 (2H, d J=8.2 Hz, Ar—H), 7.02(2H, d J=8.2 Hz, Ar—H), 4.22-4.40 (3H, m, CHCH₂Ph and CHCH₂CH(CH₃)₂ andOCH₂CH₂CH₂CH₂), 3.95-4.08 (2H, CHCO₂CH₃ and OCH₂CH₂CH₂CH₂), 3.56 (3H, s,CO₂CH₃), 3.12 (1H, dd J=5.9 Hz, J=12.7 Hz, CHCH₂Ph), 2.62 (1H, dd J=12.7Hz, J=12.7 Hz, CHCH₂Ph), 1.60-1.75 (4H, m, OCH₂CH₂CH₂CH₂ andOCH₂CH₂CH₂CH₂), 1.49-1.58 (2H, m, CHCH₂CH(CH₃)₂) 1.44-1.48 (1H, m,CHCH₂CH(CH₃)₂), 1.22-1.41 (2H, m, OCH₂CH₂CH₂CH₂), 0.85 (3H, d J=7.3 Hz,CHCH₂CH(CH₃)₂), 0.84 (3H, d J=7.3 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 419.2541 (MH⁺). C₂₃H₃₄N₂O₅ requires 419.2546.

(7S,10S,13S)-13-Benzyloxycarbonylamino-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo-[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (7)

Amine 6 (4.00 g, 8.78 mmol) was dissolved in anhydrous DMF (30 mL).Benzyl chloroformate (1.88 mL, 13.2 mmol) and DIPEA (6.11 mL, 35.1 mmol)were added and the reaction mixture was stirred at rt for 18 h beforebeing partitioned between chloroform and 1M hydrochloric acid. Theaqueous phase was extracted three more times with chloroform and thecombined organic extracts were dried (MgSO₄), filtered and concentratedin vacuo. Purification was achieved using flash chromatography, elutingwith a gradient of 15% EtOAc/DCM to 50% EtOAc/DCM to yield an off-whitesolid, 2.10 g, 43%. R_(f)=0.43 (30% EtOAc/DCM).

¹H-NMR (500 MHz in CDCl₃) 7.31-7.38 (5H, m, Ar—H (CBZ)), 7.06 (2H, dJ=7.9 Hz, Ar—H (Tyr)), 6.79 (2H, d J=7.9 Hz, Ar—H (Tyr)), 6.13 (1H, dJ=6.9 Hz, NH Gly), 5.79 (1H, d J=7.9 Hz, NH Leu), 5.53 (1H, d J=8.5 Hz,NH Tyr), 5.13 (2H, s, OCH₂Ph), 4.51-4.57 (1H, m, CHCH₂CH(CH₃)₂),4.22-4.32 (2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂), 4.11 (1H, ddd J=5.9 Hz,J=10.1 Hz, J=16.4 Hz, OCH₂CH₂CH₂CH₂), 3.95 (1H, ddd J=6.5 Hz, J=6.9 Hz,J=13.0 Hz, CHCO₂CH₃), 3.73 (3H, s, CO₂CH₃), 3.14 (1H, dd J=5.7 Hz,J=12.8 Hz, CHCH₂Ph), 2.67 (1H, dd J=12.8 Hz, J=12.8 Hz, CHCH₂Ph),1.86-1.95 (1H, m, CHCH₂CH(CH₃)₂), 1.74-1.83 (2H, m, OCH₂CH₂CH₂CH₂),1.22-1.58 (6H, m, CHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂),0.88 (3H, d J=6.0 Hz, CHCH₂CH(CH₃)₂), 0.87 (3H, d J=6.0 Hz,CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 172.5, 170.8, 169.7, 157.1, 155.5, 136.3,130.1, 128.5, 128.2, 128.1, 127.9, 115.7, 66.8, 66.7, 57.1, 52.5, 51.7,51.2, 43.3, 39.0, 31.5, 28.0, 24.5, 22.9, 22.4, 21.2.

HRMS (ES) 554.2859 (MH⁺). C₃₀H₃₉N₃O₇ requires 554.2866.

(7S,10S,13S)-7-Hydroxymethyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (8)

Methyl ester 7 (2.30 g, 4.15 mmol) was dissolved in anhydrous THF (40mL) under an atmosphere of argon. The resultant solution was cooled inice and 1M LiAlH₄ in diethyl ether was added (4.57 mL, 4.57 mmol). Thereaction mixture was stirred in ice for 1 h and then at rt for 18 h.MeOH (30 mL) was added and the reaction mixture was stirred at rt for afurther 10 mins before being concentrated in vacuo. The residue waspartitioned between EtOAc and 1M aqueous KHSO₄. The aqueous phase wasextracted twice more with chloroform and each organic extract was washedwith brine before being combined, dried (MgSO₄), filtered andconcentrated in vacuo to yield an off-white solid, 1.81 g, 83%.

¹H-NMR (500 MHz in CD₃OD) 7.68 (1H, d J=9.1 Hz, NH Gly), 7.22-7.36 (6H,m, Ar—H (CBZ) and NH Leu), 7.06 (2H, d J=7.9 Hz, Ar—H Tyr), 6.77 (2H, dJ=7.9 Hz, Ar—H Tyr), 5.10 (1H, d J=12.5 Hz, OCH₂Ph), 5.05 (1H, d J=12.1Hz, OCH₂Ph), 4.26-4.34 (2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂), 4.06-4.12(1H, m, OCH₂CH₂CH₂CH₂), 3.96-4.02 (1H, m, CHCH₂CH(CH₃)₂), 3.74-3.86 (1H,m, CHCH₂OH), 3.30-3.33 (2H, m, CH₂OH), 2.98 (1H, dd J=5.4 Hz, J=12.7 Hz,CHCH₂Ph), 2.67 (1H, dd J=12.4 Hz, J=12.7 Hz, CHCH₂Ph), 1.75-1.84 (2H, m,OCH₂CH₂CH₂CH₂), 1.46-1.56 (3H, m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂),1.22-1.44 (4H, m, OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂), 0.84 (3H, d J=8.8Hz, CHCH₂CH(CH₃)₂), 0.83 (3H, d J=8.8 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 526.2920 (MH⁺). C₂₉H₃₉N₃O₆ requires 526.2917.

(7S,10S,13S)-7-Formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9)

Alcohol 8 (1.71 g, 3.25 mmol) was dissolved in DMSO (30 mL) under anatmosphere of argon. To the resultant solution DCM (15 mL) and DIPEA(2.27 mL, 13.0 mmol) were added. The reaction mixture was cooled in iceand sulfur trioxide.pyridine complex (2.07 g, 13.0 mmol) pre-dissolvedin DMSO (15 mL) was added. This was stirred in ice for 2 h before beingpartitioned between EtOAc and 1M hydrochloric acid. The aqueous phasewas extracted again with EtOAc and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 1/2EtOAc/(50/70) petroleum ether to EtOAc to yield a light brown solid,0.720 g, 42%. R_(f)=0.41 (2/1 EtOAc/(50/70) petroleum ether).

This preparation was repeated, with purification by recrystallisationfrom MeOH, to give aldehyde 9 as white solid 1.37 g, 80%.

¹H-NMR (500 MHz in (CD₃)₂SO) 9.33 (1H, s, CHO), 8.05 (1H, d J=8.1 Hz, NHLeu), 7.55 (1H, d J=6.8 Hz, NH Tyr), 7.30-7.37 (5H, m, Ar—H CBZ), 7.16(1H, d J=8.1 Hz, NH Gly), 7.02 (2H, d J=8.0 Hz, Ar—H Tyr), 6.77 (2H, dJ=8.0 Hz, Ar—H Tyr), 5.06 (1H, d J=12.3 Hz, OCH₂Ph), 5.01 (1H, d J=12.3Hz, OCH₂Ph), 4.31-4.36 (2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂), 4.18-4.25(1H, m, CHCH₂CH(CH₃)₂), 4.00-4.07 (2H, m, CHCHO and OCH₂CH₂CH₂CH₂), 2.86(1H, dd J=5.6 Hz, J=12.8 Hz CHCH₂Ph), 2.63 (1H, dd J=12.8 Hz, J=12.8 Hz,CHCH₂Ph)), 1.70-1.77 (2H, m, OCH₂CH₂CH₂CH₂), 1.46-1.52 (1H, m,CHCH₂CH(CH₃)₂), 1.22-1.39 (6H, m, CHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂ andOCH₂CH₂CH₂CH₂, 0.80-0.83 (6H, m, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 199.1, 171.1, 155.9, 137.7, 136.4, 129.3,128.6, 128.5, 128.0, 127.9, 126.7, 66.6, 57.2, 50.2, 40.2, 38.6, 37.7,24.8, 23.0, 21.8.

HRMS (ES) 524.2762 (MH⁺). C₂₉H₃₇N₃O₆ requires 524.2760.

Reagents and Conditions: (i) HATU, DIPEA, Leu-OMe, DMF, (80%); (ii) 4MHCl, 1,4-dioxane, (100%); (iii) 4-fluorobenzene sulfonyl chloride,DIPEA, DCM, (65%); (iv) NaOH, THF, H₂O, MeOH, (95%); (v) HATU, DIPEA,(s)-allyl-Gly-OMe, DMF, (89%); (vi) 3×10 mol % GSGC, 10 mol %chloro-dicyclohexyl borane, 1,1,2-TCE, microwave, (65%); (vii) H₂, 20mol % Pd/C, MeOH, EtOAc, (93%); (viii) LiAlH₄, THF, (86%) (ix) SO₃.Pyr,DIPEA, DMSO, DCM (69%).

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-amino-propionylamino]-4-methyl-pentanoicacid methyl ester hydrogen chloride salt (10)

Methyl ester 1 (4.00 g, 8.92 mmol) was dissolved in 4M HCl in1,4-dioxane (20 mL). The resultant solution was stirred at rt for 18 hbefore being concentrated in vacuo to yield a white solid, 3.43 g, 100%.

¹H-NMR (500 MHz in CDCl₃) 8.28 (2H, bs, NH₂), 7.67 (1H, d J=6.4 Hz NHLeu), 7.27 (2H, d J=8.4 Hz, Ar—H), 6.82 (2H, d J=8.4 Hz, Ar—H), 6.00(1H, tdd J=5.2 Hz, J=5.2 Hz, J=10.3 Hz, J=16.9 Hz, CH₂CHCH₂), 5.22-5.40(2H, m, CH₂CHCH₂), 4.57-4.64 (1H, m, CHCH₂Ph), 4.44 (2H, d J=5.2 Hz,OCH₂CHCH₂), 4.28-4.37 (1H, m, CHCH₂CH(CH₃)₂), 3.63 (3H, s, CO₂CH₃), 3.36(1H, dd J=4.3 Hz, J=13.9 Hz, CHCH₂Ph), 3.20 (1H, dd J=8.2 Hz, J=13.9 Hz,CHCH₂Ph), 1.53-1.63 (3H, m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 0.83 (3H,d J=6.7 Hz, CHCH₂CH(CH₃)₂), 0.82 (3H, d J=6.7 Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 172.4, 168.2, 157.9, 133.1, 131.0, 126.2,117.5, 114.9, 68.6, 67.0, 54.6, 52.2, 51.4, 40.1, 36.2, 24.5, 22.5,21.9.

LRMS (ES) 349.2 (MH⁺). C₁₉H₂₈N₂O₆ requires 349.2.

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-propionylamino]-4-methyl-pentanoicacid methyl ester (11)

Amine 10 (3.50 g, 9.10 mmol) and 4-fluoro-benzenesulfonyl chloride (1.61g, 8.27 mmol) were dissolved in anhydrous DCM (40 mL). DIPEA (3.17 mL,18.2 mmol) was added and the reaction mixture was stirred at rt for 18 hbefore being concentrated in vacuo. The residue was partitioned betweenEtOAc and 1M hydrochloric acid. The organic phase was washedsequentially with 1M hydrochloric acid and brine before being dried(MgSO₄), filtered and concentrated in vacuo. Purification was achievedusing flash chromatography, eluting with a gradient of (50/70) petroleumether to 2/1 (EtOAc/(50/70) petroleum ether to yield a white solid, 2.70g, 65%. R_(f)=0.81 (1/1 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.68-7.71 (2H, m, Ar—H (4-F-Ph)), 7.06-7.11(2H, m, Ar—H (4-F-Ph)), 6.91 (2H, d J=8.6 Hz, Ar—H (Tyr)), 6.73 (2H, dJ=8.6 Hz, Ar—H (Tyr)), 6.42 (1H, d J=8.2 Hz, NH Leu), 6.05 (1H, tddJ=5.3 Hz, J=5.3 Hz, J=10.5 Hz, J=17.2 Hz, CH₂CHCH₂) 5.27-5.44 (2H, m,CH₂CHCH₂), 4.46-4.51 (3H, m, OCH₂CHCH₂, and CHCH₂CH(CH₃)₂), 3.84-3.88(1H, m, CHCH₂Ph), 3.71 (3H, s, CO₂CH₃), 2.94 (1H, dd J=7.2 Hz, J=14.4Hz, CHCH₂Ph), 2.90 (1H, dd J=6.4 Hz, J=14.4 Hz, CHCH₂Ph) 1.52-1.58 (1H,m, CHCH₂CH(CH₃)₂) 1.38-1.47 (2H, m, CHCH₂CH(CH₃)₂) 0.88 (6H, d J=6.2 Hz,CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 172.5, 169.6, 157.9, 133.0, 130.2, 129.9,129.8, 127.0, 117.8, 116.4, 116.1, 115.0, 68.7, 57.8, 52.3, 50.9, 41.4,37.8, 24.6, 22.6, 21.8.

LRMS (ES) 507.3 (MH⁺). C₂₅H₃₁FN₂O₆S requires 507.2.

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-propionylamino]-4-methyl-pentanoicacid (12)

Methyl ester 11 (2.70 g, 5.33 mmol) was dissolved in THF (30 mL). Sodiumhydroxide (0.853 g, 21.3 mmol) pre-dissolved in water (10 mL) was added.Methanol (15 mL) was added to obtain a homogenous solution and this wasstirred at rt for 18 h before being concentrated in vacuo. The residuewas partitioned between EtOAc and 1M hydrochloric acid. The aqueousphase was extracted twice more with EtOAc and the combined organicextracts were washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo to yield a white foam, 2.50 g, 95%.

¹H-NMR (500 MHz in CD₃OD) 8.27 (1H, d, J=8.0 Hz, NH Leu), 7.60-7.63 (2H,m, Ar—H (4-F-Ph)), 7.04-7.08 (2H, m, Ar—H (4-F-Ph)) 7.02 (2H, d J=8.5Hz, Ar—H Tyr) 6.71 (2H, d J=8.5 Hz Ar—H Tyr) 6.04 (1H, tdd J=5.2 Hz,J=5.2 Hz, J=10.5 Hz, J=17.3 Hz, OCH₂CHCH₂) 5.20-5.40 (2H, m, OCH₂CHCH₂),4.49 (2H, d J=5.2 Hz, OCH₂CHCH₂), 4.22-4.27 (1H, m, CHCH₂CH(CH₃)₂), 3.99(1H, dd J=4.5 Hz, J=9.5 Hz, CHCH₂Ph), 2.96 (1H, dd J=4.5 Hz, J=14.0 Hz,CHCH₂Ph), 2.65 (1H, dd J=9.5 Hz, J=14.0 Hz, CHCH₂Ph), 1.45-1.56 (3H, m,CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 0.90 (3H, d J=5.8 Hz, CHCH₂CH(CH₃)₂),0.84 (3H, d J=5.8 Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 174.2, 172.0, 166.4, 157.6, 137.0, 133.7,130.8, 130.1, 129.6, 129.4, 128.7, 116.1, 115.7, 115.4, 114.1, 68.4,58.0, 50.6, 40.4, 37.9, 24.5, 22.0, 20.6.

LRMS (ES) 493.2 (MH⁺). C₂₄H₂₉FN₂O₆S requires 493.2.

(S)-2-{(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-propionylamino]-4-methyl-pentanoylamino}-pent-4-enoicacid methyl ester (13)

Carboxylic acid 12 (2.50 g, 5.08 mmol), HATU (2.13 g, 5.59 mmol) and(S)-allyl-glycine methyl ester hydrochloride (0.925 g, 5.59 mmol) weredissolved in DMF (30 mL). DIPEA was added (3.54 mL, 20.3 mmol) and thereaction mixture was stirred at rt for 18 h before being partitionedbetween EtOAc and 1M hydrochloric acid. The organic phase was washedsequentially with 1M hydrochloric acid and brine before being dried(MgSO₄), filtered and concentrated in vacuo. Purification was achievedusing flash chromatography, eluting with a gradient of (50/70) petroleumether to 2/1 EtOAc/(50/70) petroleum ether to yield a white solid, 2.70g, 89%. R_(f)=0.38 (1/1 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CD₃OD) 7.61-7.65 (2H, m, Ar—H (4-F-Ph)), 7.06-7.10(2H, m, Ar—H (4-F-Ph), 6.97 (2H, d J=8.5 Hz, Ar—H Tyr), 6.69 (2H, dJ=8.5 Hz, Ar—H Tyr), 6.05 (1H, tdd J=5.2 Hz, J=5.2 Hz, J=10.5 Hz, J=17.3Hz, OCH₂CHCH₂), 5.78 (1H, tdd J=7.1 Hz, J=10.2 Hz, J=17.1 Hz,CHCH₂CHCH₂), 5.04-5.42 (4H, m, OCH₂CHCH₂ and CHCH₂CHCH₂), 4.48 (2H, dJ=5.2 Hz, OCH₂CHCH₂), 4.42 (1H, dd J=5.6 Hz, J=8.0 Hz, CHCO₂CH₃), 4.32(1H, dd J=5.0 Hz, J=9.4 Hz, CHCH₂CH(CH₃)₂), 3.95 (1H, dd J=4.7 Hz, J=9.5Hz, CHCH₂Ph), 3.69 (3H, s, CO₂CH₃), 2.93 (1H, dd J=4.7 Hz, J=14.0 Hz,CHCH₂Ph), 2.63 (1H, dd J=9.5 Hz, J=14.0 Hz, CHCH₂Ph), 2.41-2.57 (2H, m,CHCH₂CHCH₂), 1.50-1.57 (2H, m, CHCH₂CH(CH₃)₂), 1.44-1.48 (1H, m,CHCH₂CH(CH₃)₂), 0.92 (3H, d J=6.0 Hz, CHCH₂CH(CH₃)₂), 0.87 (3H, d J=6.0Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 171.7, 171.5, 170.4, 167.3, 163.7, 157.7,134.4, 134.3, 133.0, 132.3, 130.2, 129.9, 129.8, 127.2, 118.9, 117.7,116.3, 116.0, 114.8, 68.6, 58.4, 52.2, 51.9, 51.9, 41.0, 37.5, 36.1,24.6, 22.8, 21.9.

LRMS (ES) 604.3 (MH⁺). C₃₀H₃₈FN₃O₇S requires 604.2.

(E/Z)-(7S,10S,13S)-13-(4-Fluoro-benzenesulfonylamino)-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),4,15(19),16-tetraene-7-carboxylicacid methyl ester (14)

Diene 13 (1.18 g, 1.95 mmol) was dissolved in 1,1,2-trichloroethane (190mL) under an atmosphere of argon. Chloro-dicyclohexyl borane (0.195 mL,0.195 mmol) and GSGC (0.166 g, 0.195 mmol) were added. The reactionmixture was heated at reflux in the microwave (1200 W) for 20 mins. Twofurther additions of GSGC (0.166 g, 0.195 mmol) were added and aftereach the reaction mixture was subjected to a further 20 mins heating inthe microwave. The reaction mixture was then cooled and concentrated invacuo. Purification was achieved using flash chromatography eluting witha gradient of (50/70) petroleum ether to 100% EtOAc to yield a brownsolid, 0.700 g, 63%. The product was obtained as a mixture of E/Zisomers. R_(f)=0.17 (1/1 EtOAc/(50/70) petroleum ether).

¹H-NMR for major isomer from mixture (500 MHz in (CD₃)₂SO) 8.18 (1H, dJ=7.6 Hz, NH Tyr), 8.08 (1H, d J=8.3 Hz, NH Gly), 7.88-7.93 (2H, m, Ar—H(4-F-Ph)), 7.58 (1H, d J=7.5 Hz, NH Leu), 7.32-7.38 (2H, m, Ar—H(4-F-Ph)), 6.90 (2H, d J=7.7 Hz, Ar—H (Tyr)), 6.64 (2H, d J=7.7 Hz, Ar—H(Tyr)), 5.52-5.56 (1H, m, OCH₂CHCHCH₂), 5.39-5.46 (1H, m, OCH₂CHCHCH₂),4.53-4.67 (2H, m, OCH₂CHCHCH₂), 4.20-4.30 (2H, m, CHCH₂Ph and CHCO₂CH₃),3.78-3.85 (1H, m, CHCH₂CH(CH₃)₂), 3.54 (3H, s, CO₂CH₃), 2.58-2.72 (2H,m, CHCH₂Ph), 2.14-2.25 (2H, m, OCH₂CHCHCH₂), 1.15-1.25 (2H, m,CHCH₂CH(CH₃)₂), 0.99-1.05 (1H, m, CHCH₂CH(CH₃)₂), 0.75 (3H, d J=6.8 Hz,CHCH₂CH(CH₃)₂), 0.70 (3H, d J=6.8 Hz, CHCH₂CH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 7.15-7.22 (2H, m, Ar—H(4-F-Ph)), 7.02-7.06 (2H, m, Ar—H (Tyr)), 0.73 (3H, d J=6.8 Hz,CHCH₂CH(CH₃)₂), 0.69 (3H, d J=6.8 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 576.2191 (MH⁺). C₂₈H₃₄FN₃O₇S requires 576.2180.

(7S,10S,13S)-13-(4-Fluoro-benzenesulfonylamino)-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (15)

Olefin 14 (0.700 g, 1.22 mmol) was dissolved in a mixture of DCM (30mL), EtOAc (10 mL) and MeOH (10 mL). 10% palladium on carbon catalystwas added (0.140 g, 20%). The reaction mixture was subjected tohydrogenation at rt and atmospheric pressure for 18 h before beingfiltered through celite and concentrated in vacuo to yield a brownsolid, 0.667 g, 95%.

¹H-NMR (500 MHz in (CD₃)₂SO) 8.11-8.15 (2H, m, NH Tyr and NH Gly),7.91-7.95 (1H, m, NH Leu), 7.92-7.96 (2H, m, Ar—H (4-F-Ph)), 7.33-7.37(2H, m, Ar—H (4-F-Ph)), 6.92-6.97 (2H, m, Ar—H (Tyr)), 6.70 (2H, d J=7.9Hz, Ar—H (Tyr)), 4.28-4.38 (2H, m, CHCOCH₃ and OCH₂CH₂CH₂CH₂), 4.11-4.21(2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂), 3.95-4.04 (1H, m, CHCH₂CH(CH₃)₂),3.52 (3H, s, CO₂CH₃), 2.69-2.75 (1H, m, CHCH₂Ph), 2.54 (1H, dd J=7.1 Hz,J=12.0 Hz, CHCH₂Ph), 1.73-1.78 (2H, m, OCH₂CH₂CH₂CH₂), 1.20-1.70 (7H, m,OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂),0.73 (3H, d J=6.3 Hz, CHCH₂CH(CH₃)₂), 0.72 (3H, d J=6.3 Hz,CHCH₂CH(CH₃)₂).

HRMS (ES) 578.2337 (MH⁺). C₂₈H₃₆FN₃O₇S requires 578.2336.

4-Fluoro-N-((7S,10S,13S)-7-hydroxymethyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo-[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-benzenesulfonamide(16)

Methyl ester 15 (0.677 g, 1.17 mmol) was dissolved in anhydrous THF (30mL) under an atmosphere of argon. The resultant solution was cooled inice and 1M LiAlH₄ in diethyl ether was added (1.17 mL, 1.17 mmol). Thereaction mixture was stirred in ice for 1 h and then at rt for 18 h.MeOH (30 mL) was added and the reaction mixture was stirred at rt for afurther 10 mins before being concentrated in vacuo. The residue waspartitioned between EtOAc and 1M aqueous KHSO₄. The aqueous phase wasextracted twice more with chloroform and each organic extract was washedwith brine before being combined, dried (MgSO₄), filtered andconcentrated in vacuo to yield a brown solid, 0.550 g, 92%.

¹H-NMR (500 MHz in CD₃OD) 7.95-7.99 (2H, m, Ar—H (4-F-Ph)), 7.67 (1H, dJ=8.8 Hz, NH Leu), 7.25-7.28 (2H, m, Ar—H (4-F-Ph)), 7.17 (1H, d J=7.5Hz, NH Gly), 6.97 (2H, d J=8.0 Hz, Ar—H (Tyr)), 6.76 (2H, d J=8.0 Hz,Ar—H (Tyr)), 4.29 (1H, m, OCH₂CH₂CH₂CH₂), 4.08 (2H, m, CHCH₂Ph andOCH₂CH₂CH₂CH₂), 3.85-3.87 (2H, m, CHCH₂OH and CHCH₂CH(CH₃)₂), 3.24-3.33(2H, m, CH₂OH), 2.88 (1H, dd J=5.5 Hz, J=12.3 Hz, CHCH₂Ph), 2.69 (1H, ddJ=12.3 Hz, J=12.3 Hz, CHCH₂Ph), 1.72-1.81 (2H m, OCH₂CH₂CH₂CH₂),1.22-1.68 (7H, m, OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂ andCHCH₂CH(CH₃)₂), 0.78 (3H, d J=6.4 Hz, CHCH₂CH(CH₃)₂), 0.76 (3H, d J=6.4Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CD₃OD). 171.6, 169.7, 166.6, 163.3, 157.1, 130.0,129.8, 129.6, 127.7, 116.1, 115.8, 66.7, 64.4, 57.9, 51.8, 50.1, 43.2,39.0, 29.7, 28.3, 24.2, 22.2, 22.1, 21.4.

HRMS (ES) 550.2369 (MH⁺). C₂₇H₃₆FN₃O₆S requires 550.2387.

4-Fluoro-N-((7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,1′-diaza-bicyclo[13.2.2]-nonadeca-1(18),15(19),16-trien-13-yl)-benzenesulfonamide(17)

Alcohol 16 (0.57 g, 1.14 mmol) was dissolved in DMSO (12 mL) under anatmosphere of argon. To the resultant solution DCM (6 mL) and DIPEA(1.20 mL, 5.13 mmol) were added. The reaction mixture was cooled in iceand sulfur trioxide.pyridine complex (0.78 g, 4.90 mmol) pre-dissolvedin DMSO (6 mL) was added. Stirring was continued in ice for 2 h beforethe reaction mixture was partitioned between EtOAc and 1M hydrochloricacid. The aqueous phase was extracted again with EtOAc and the combinedorganic extracts were dried (MgSO₄), filtered and concentrated in vacuo.Purification was achieved using flash chromatography, eluting with agradient of (50/70) petroleum ether to EtOAc to yield a light brownsolid, 0.165 g, 29%. R_(f)=0.28 (2/1 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in (CD₃)₂SO) 9.29 (1H, s, CHO), 8.15 (1H, d J=8.9 Hz, NHTyr), 8.04 (1H, d J=8.3 Hz, NH Leu), 7.93-7.97 (2H, m, Ar—H (4-F-Ph)),7.49 (1H, d J=7.8 Hz, NH Gly), 7.33-7.38 (2H, m, Ar—H (4-F-Ph)), 6.95(2H, d J=7.6 Hz, Ar—H (Tyr)), 6.73 (2H, d J=7.6 Hz, Ar—H (Tyr)),4.22-4.37 (2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂), 4.17-4.24 (1H, m,CHCH₂CH(CH₃)₂), 3.97-4.04 (1H, m, OCH₂CH₂CH₂CH₂), 3.80-3.87 (1H, m,CHCHO), 2.72 (1H, dd J=5.2 Hz, J=12.7 Hz, CHCH₂Ph), 2.54-2.60 (1H, m,CHCH₂Ph), 1.32-1.74 (7H, m, OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂ andCHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂) 0.70-0.75 (6H, m, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in (CD₃)₂SO). 201.0, 171.0, 168.4, 155.9, 130.3, 129.8,129.7, 127.8, 116.1, 115.8, 115.5, 66.0, 56.5, 56.3, 50.5, 43.2, 26.8,26.3, 23.8, 23.1, 22.3, 21.4.

HRMS (ES) 548.2226 (MH⁺). C₂₇H₃₄FN₃O₆S requires 548.2230.

Reagents and Conditions: (i) K₂CO₃, 4-bromo-but-1-ene, DMF, (27%); (ii)NaOH, THF, H₂O, MeOH, (85%); (iii) HATU, DIPEA, Leu-OMe, DMF, (64%);(iv) NaOH, THF, H₂O, MeOH, (96%); (v) HATU, DIPEA, (s)-allyl-Gly-OMe,DMF, (84%); (vi) 3×10 mol % GSGC, 10 mol % chlorodicyclohexylborane,1,1,2-TCE, microwave, (100%); (vii) H₂, 20 mol % Pd/C, MeOH, EtOAc,(26%) (viii) 4M HCl, 1,4-dioxane (100%); (ix) benzyl chloroformate,DIPEA, DMF, (69%); (x) LiAlH₄, THF, (72%); (xi) SO₃.Pyr, DIPEA, DMSO,DCM, (82%).

(S)-3-(4-But-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acidmethyl ester (18)

N—BOC-tyrosine methyl ester (10.0 g, 33.9 mmol) was dissolved in DMF (40mL) and potassium carbonate (5.62 g, 40.7 mmol) and 4-bromo-1-butene(4.13 mL, 40.7 mmol) were added. The mixture was stirred at rt for 18 hbefore being diluted with EtOAc (120 mL) and partitioned with 1Mhydrochloric acid. The organic phase was washed with hydrochloric acidand then with brine before being dried (MgSO₄), filtered andconcentrated in vacuo. The crude material was purified by flashchromatography on silica using a gradient of EtOAc and (50/70) petroleumether to yield a white solid, 3.20 g, 27%. R_(f)=0.82 (1/2 EtOAc/(50/70)petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.01 (2H, d J=6.3 Hz, Ar—H), 6.82 (2H, d J=6.3Hz, Ar—H), 5.85-5.94 (1H, m, OCH₂CH₂CHCH₂), 5.06-5.31 (2H, m,OCH₂CH₂CHCH₂), 4.95 (1H, d J=6.1 Hz, NH), 4.48-4.58 (1H, m, CHCO₂CH₃),3.98 (2H, t J=6.4 Hz, J=6.7 Hz, J=6.7 Hz, OCH₂CH₂CHCH₂), 3.70 (3H, s,CHCO₂CH₃), 3.04 (1H, dd J=6.0 Hz, J=13.9 Hz, CHCH₂Ph), 2.98 (1H, ddJ=5.0 Hz, J=13.9 Hz, CHCH₂Ph), 2.53 (2H, dt J=3.2 Hz, J=6.4 Hz, J=8.2Hz, OCH₂CH₂CHCH₂), 1.41 (9H, s, C(CH₃)₃).

HRMS (ES) 350.1975 (MH⁺). C₁₉H₂₇NO₅ requires 350.1967.

(S)-3-(4-But-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acid(19)

Methyl ester 18 (3.20 g, 9.16 mmol) was dissolved in THF (30 mL) andNaOH (0.552 g, 13.8 mmol) dissolved in water (10 mL) was added. Methanol(10 mL) was added to obtain a homogenous solution and this was stirredat rt for 18 h. The reaction mixture was then concentrated in vacuo andthe residue was partitioned between EtOAc and 1M hydrochloric acid. Theaqueous phase was extracted twice more with EtOAc and the combinedorganic extracts were washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo to yield a white solid, 2.60 g, 85%. m.p. 148-150°C.

¹H-NMR (500 MHz in CD₃OD) 7.11 (2H, d J=7.3 Hz, Ar—H), 6.78 (2H, d J=7.3Hz, Ar—H), 5.91 (1H, ddt J=2.0 Hz, J=6.7 Hz, J=17.0 Hz, OCH₂CH₂CHCH₂),5.05-5.16 (2H, m, OCH₂CH₂CHCH₂), 4.16-4.19 (1H, m, CHCO₂H), 3.97 (2H, tJ=6.3 Hz, OCH₂CH₂CHCH₂), 3.09 (1H, dd J=3.9 Hz, J=13.7 Hz, CHCH₂Ph),2.84 (1H, dd J=7.6 Hz, J=13.9 Hz, CHCH₂Ph), 2.48-2.51 (2H, m,OCH₂CH₂CHCH₂), 1.38 (9H, s, C(CH₃)₃).

LRMS (ES) 358.2 (MNa⁺). C₁₈H₂₅NO₅Na requires 358.2.

(S)-2-[(S)-3-[(4-But-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-ethyl-pentanoicacid methyl ester (20)

Carboxylic acid 19 (2.60 g, 7.75 mmol), HATU (3.24 g, 8.53 mmol) andleucine methyl ester hydrochloride (2.82 g, 15.5 mmol) were dissolved inDMF (40 mL). DIPEA was added (5.40 mL, 31.0 mmol) and the reactionmixture was stirred at rt for 18 h. before being partitioned betweenEtOAc and 1M hydrochloric acid. The organic phase was washedsequentially with 1M hydrochloric acid and brine before being dried(MgSO₄), filtered and concentrated in vacuo. The crude material waspurified by flash chromatography on silica using a gradient of EtOAc and(50/70) petroleum ether to yield a white solid, 2.30 g, 64%. R_(f)=0.34(1/2 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.11 (2H, d J=8.5 Hz, Ar—H), 6.82 (2H, d J=8.5Hz, Ar—H), 6.24 (1H, d J=7.5 Hz, NH Leu), 5.85-5.94 (1H, m,OCH₂CH₂CHCH₂), 5.08-5.17 (2H, m, OCH₂CH₂CHCH₂), 5.01 (1H, bs, NH Tyr),4.54-4.58 (1H, m, CHCH₂CH(CH₃)₂), 4.27-4.32 (1H, m, CHCH₂Ph), 3.98 (2H,dt J=2.3 Hz, J=6.7 Hz, OCH₂CH₂CHCH₂), 3.69 (3H, s, CO₂CH₃), 2.95-3.05(2H, m, CHCH₂Ph), 2.51-2.55 (2H, m, OCH₂CH₂CHCH₂), 1.53-1.60 (2H, m,CHCH₂CH(CH₃)₂), 1.44-1.49 (1H, m, CHCH₂CH(CH₃)₂), 1.42 (9H, s, C(CH₃)₃),0.88-0.91 (6H, m, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 172.8, 171.0, 157.8, 134.4, 130.3, 130.2,128.4, 116.9, 114.6, 67.1, 55.7, 52.2, 50.6, 41.5, 37.1, 33.6, 28.2,24.6, 22.7, 21.8.

HRMS (ES) 463.2809 (MH⁺). C₂₅H₃₈N₂O₆ requires 463.2808.

(S)-2-[(S)-3-(4-But-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-methyl-pentanoicacid (21)

Methyl ester 20 (2.30 g, 4.97 mmol) was dissolved in THF (30 mL) andNaOH (0.298 g, 7.46 mmol) dissolved in water (10 mL) was added. Methanol(10 mL) was added to obtain a homogenous solution and this was stirredat rt for 18 h. The reaction mixture was then concentrated in vacuo andthe residue was partitioned between EtOAc and 1M hydrochloric acid. Theaqueous phase was extracted twice more with EtOAc and the combinedorganic extracts were washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo to yield a white solid, 2.14 g, 96%. m.p. 68-70°C.

¹H-NMR (500 MHz in CDCl₃) 9.41 (1H, bs, CO₂H), 7.09 (2H, d J=8.1 Hz,Ar—H), 6.80 (2H, d J=8.1 Hz, Ar—H), 6.70 (1H, d J=7.7 Hz, NH Leu),5.84-5.92 (1H, m, OCH₂CH₂CHCH₂), 5.29 (1H, bs, NH Tyr), 5.07-5.16 (2H,m, OCH₂CH₂CHCH₂), 4.54-4.57 (1H, m, CHCH₂CH(CH₃)₂), 4.36-4.37 (1H, m,CHCH₂Ph), 3.95 (2H, t J=6.5 Hz, J=6.5 Hz, OCH₂CH₂CHCH₂), 2.93-3.02 (2H,m, CHCH₂Ph), 2.49-2.51 (2H, m, OCH₂CH₂CHCH₂) 1.50-1.69 (3H, m,CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 1.38 (9H, s, C(CH₃)₃), 0.90-0.92 (6H,m, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 175.9, 171.6, 157.8, 134.4, 130.3, 130.2,128.4, 116.9, 114.6, 67.1, 55.6, 50.8, 41.1, 37.7, 37.1, 30.6, 28.2,24.6, 22.8, 21.8.

LRMS (ES) 449.6 (MH⁺). C₂₄H₃₆N₂O₆ requires 449.3.

Microanalysis: C, 64.36; H, 7.92; N, 6.15. C₂₄H₃₆N₂O₆ requires C, 64.26;H, 8.09; N, 6.25.

(S)-2-{(S)-2-[(S)-3-(4-But-3-enyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-4-methyl-pentanoylamino}-pent-4-enoicacid methyl ester (22)

Carboxylic acid 21 (2.14 g, 4.77 mmol), HATU (2.00 g, 5.25 mmol) and(S)-allyl-glycine methyl ester hydrochloride (0.870 g, 5.25 mmol) weredissolved in DMF (30 mL). DIPEA was added (3.32 mL, 19.1 mmol) and thereaction mixture was stirred at rt for 18 h before being partitionedbetween EtOAc and 1M hydrochloric acid. The organic phase was washedsequentially with 1M hydrochloric acid and brine before being dried(MgSO₄), filtered and concentrated in vacuo. The crude material waspurified by flash chromatography on silica using a gradient of EtOAc and(50/70) petroleum ether to yield a white solid, 2.24 g, 84%. R_(f)=0.32(1/1 EtOAc/(50/70) petroleum ether). m.p. 166-168° C.

¹H-NMR (500 MHz in CDCl₃) 7.09 (2H, d J=8.4 Hz, Ar—H), 6.93 (1H, d J=7.7Hz, NH Gly), 6.80 (2H, d J=8.4 Hz, Ar—H), 6.71 (1H, d J=7.3 Hz, NH Leu),5.89 (1H, tdd J=6.7 Hz, J=10.1 Hz, J=17.0 Hz, OCH₂CH₂CHCH₂), 5.68-5.72(1H, m, CHCH₂CHCH₂), 5.09-5.22 (4H, m, OCH₂CH₂CHCH₂ and CHCH₂CHCH₂),4.54-4.60 (1H, m, CHCO₂CH₃), 4.50 (1H, ddd J=6.0 Hz, J=6.0 Hz, J=7.3 Hz,CHCH₂CH(CH₃)₂), 4.36-4.38 (1H, m, CHCH₂Ph), 3.96 (2H, t J=6.7 Hz,OCH₂CH₂CHCH₂), 3.73 (3H, s, CHCO₂CH₃), 2.99 (1H, dd J=6.0 Hz, J=14.0 Hz,CHCH₂Ph), 2.93 (1H, dd J=6.9 Hz, J=14.0 Hz, CHCH₂Ph), 2.45-2.58 (4H, m,CHCH₂CHCH₂ and OCH₂CH₂CHCH₂, 1.54-1.66 (2H, m, CHCH₂CH(CH₃)₂), 1.43-1.48(1H, m, CHCH₂CH(CH₃)₂), 1.39 (9H, s, C(CH₃)₃), 0.86 (3H, d J=6.3 Hz,CHCH₂CH(CH₃)₂), 0.86 (3H, d J=6.3 Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 171.8, 171.6, 171.5, 157.8, 134.4, 132.2,130.3, 130.2, 128.6, 118.9, 116.9, 114.5, 79.8, 67.0, 55.6, 52.2, 51.8,51.5, 41.1, 37.2, 36.3, 36.2, 33.6, 28.2, 24.4, 22.8, 22.2.

HRMS (ES) 560.3346 (MH⁺). C₃₀H₄₅N₃O₇ requires 560.3336.

(E/Z)-(8S,11S,14S)-14-tert-Butoxycarbonylamino-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo[14.2.2]icosa-1(19),5,16(20),17-tetraene-8-carboxylicacid methyl ester (23)

Diene 22 (2.20 g, 3.93 mmol) was dissolved in 1,1,2-trichloroethane (200mL) under an atmosphere of argon. Chloro-dicyclohexyl borane (0.393 mL,0.393 mmol) and GSGC (0.334 g, 0.393 mmol) were added. The reactionmixture was heated at reflux in the microwave (1200 W) for 20 mins. Twofurther additions of GSGC (0.334 g, 0.393 mmol) were added and aftereach the reaction mixture was subjected to a further 20 mins heating inthe microwave. The resultant solution was allowed to cool before beingconcentrated in vacuo. The crude material was purified by flashchromatography on silica using a gradient of EtOAc and (50/70) petroleumether to yield a brown solid, 2.09 g, 100%. A 1:1.9 ratio of geometricisomers was obtained. R_(f)=0.42 and 0.43 (1/1 EtOAc/(50/70) petroleumether). m.p. 131-133° C.

¹H-NMR for major isomer from mixture (500 MHz in CDCl₃) 7.09 (2H, dJ=8.1 Hz, Ar—H), 6.75 (2H, d J=8.1 Hz, Ar—H), 6.02 (1H, d J=7.8 Hz, NHLeu), 5.96 (1H, d J=8.0 Hz, NH Gly), 5.40-5.65 (2H, m, OCH₂CHCHCH₂CH₂and OCH₂CHCHCH₂CH₂), 5.24 (1H, d J=8.3 Hz, NH Tyr), 4.93 (1H, ddd J=1.2Hz, J=6.2 Hz, J=13.3 Hz, OCH₂CHCHCH₂CH₂), 4.58-4.77 (1H, m,OCH₂CHCHCH₂CH₂), 4.45 (1H, ddd J=3.4 Hz, J=8.6 Hz, J=8.8 Hz, CHCO₂CH₃),4.10-4.32 (2H, m, CHCH₂Ph, CHCH₂CH(CH₃)₂), 3.74 (3H, s, CHCO₂CH₃), 3.01(1H, dd J=4.6 Hz, J=12.8 Hz, CHCH₂Ph), 2.82-2.86 (1H, m, CHCH₂Ph),2.37-2.48 (2H, m, OCH₂CHCHCH₂CH₂), 2.20-2.36 (2H, m, OCH₂CHCHCH₂CH₂),1.47-1.60 (3H, m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 1.45 (9H, s,C(CH₃)₃), 0.84-0.90 (6H, m, CHCH₂CH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 7.12 (2H, d J=8.4 Hz,Ar—H), 6.79 (2H, d J=8.4 Hz, Ar—H), 6.20 (1H, d J=7.4 Hz, NH Leu), 6.15(1H, d J=8.6 Hz, NH Gly), 5.73 (1H, d J=7.6 Hz, NH Tyr).

HRMS (ES) 532.3034 (MH⁺). C₂₈H₄₁N₃O₇ requires 532.3023.

(8S,11S,14S)-14-tert-Butoxycarbonylamino-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo-[14.2.2]icosa-1(19),16(20),17-triene-8-carboxylicacid methyl ester (24)

Olefin 23 (2.30 g, 4.33 mmol) was dissolved in a mixture of DCM (50 mL)and MeOH (50 mL). 10% palladium on carbon catalyst was added (0.460 g,20%) and the reaction mixture was subjected to hydrogenation at rt andatmospheric pressure for 18 h before being filtered through celite andconcentrated in vacuo to yield a brown solid, 0.600 g, 26%. m.p.198-200° C.

¹H-NMR (500 MHz in CD₃OD) 8.13 (1H, d J=7.6 Hz, NH Leu), 7.44 (1H dJ=8.3 Hz, NH Gly), 7.04 (2H, d J=8.5 Hz, Ar—H), 6.73 (2H, d J=8.5 Hz,Ar—H), 4.20-4.27 (2H, m, CHCO₂CH₃ and CHCH₂CH(CH₃)₂), 4.11-4.20 (2H, m,OCH₂CH₂CH₂CH₂CH₂ and CHCH₂Ph), 3.94-3.99 (1H, m, OCH₂CH₂CH₂CH₂CH₂), 3.65(3H, s, CHCO₂CH₃), 2.93 (1H, dd J=4.9 Hz, J=12.6 Hz, CHCH₂Ph), 2.71 (1H,dd J=11.8 Hz, J=12.6 Hz, CHCH₂Ph), 1.63-1.74 (3H, m, OCH₂CH₂CH₂CH₂CH₂and OCH₂CH₂CH₂CH₂CH₂), 1.51-1.60 (3H, m, CHCH₂CH(CH₃)₂ andCHCH₂CH(CH₃)₂), 1.43 (9H, s, C(CH₃)₃), 1.21-1.37 (5H, m,OCH₂CH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂CH₂), 0.86 (6H, dJ=6.4 Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CD₃OD) 172.7, 172.1, 171.4, 157.4, 156.0, 130.0,128.1, 114.8, 79.2, 66.3, 56.3, 51.4, 51.3, 42.6, 37.0, 31.2, 27.4,27.3, 24.1, 23.9, 22.1, 21.6.

HRMS (ES) 534.3107 (MH⁺). C₂₈H₄₃N₃O₇ requires 534.3179.

FTIR (KBr) 3302, 2929, 1944, 1747, 1741, 1685, 1647, 1541, 1508, 1248.

(8S,11S,14S)-14-Amino-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo[14.2.2]icosa-1(19),16(20),17-triene-8-carboxylicacid methyl ester hydrochloride (25)

Methyl ester 24 (0.600 g, 1.12 mmol) was dissolved in 4M-HCl in1,4-dioxane (20 mL). The resultant solution was stirred at rt for 18 hbefore being concentrated in vacuo to yield an off white solid, 0.528 g,100%.

¹H-NMR (500 MHz in CD₃OD) 7.04 (2H, d J=8.1 Hz, Ar—H), 6.79 (2H, d J=8.1Hz, Ar—H), 4.24-4.27 (1H, m, CHCO₂CH₃), 4.11-4.20 (2H, m,OCH₂CH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂), 4.07 (1H, dd J=4.7 Hz, J=10.9 Hz,CHCH₂Ph), 3.96-4.01 (1H, m, OCH₂CH₂CH₂CH₂CH₂), 3.64 (3H, s, CHCO₂CH₃),3.16 (1H, dd J=4.7 Hz, J=12.5 Hz, CHCH₂Ph), 2.81 (1H, dd J=10.9 Hz,J=12.5 Hz, CHCH₂Ph), 1.69-1.79 (2H m, OCH₂CH₂CH₂CH₂CH₂), 1.49-1.61 (3H,m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 1.34-1.47 (2H, m,OCH₂CH₂CH₂CH₂CH₂), 1.25-1.33 (4H, m, OCH₂CH₂CH₂CH₂CH₂ andOCH₂CH₂CH₂CH₂CH₂), 0.90 (3H, d J=6.4 Hz, CHCH₂CH(CH₃)₂), 0.88 (3H, dJ=6.4 Hz, CHCH₂CH(CH₃)₂).

¹³C-NMR (75 MHz in CD₃OD) 172.7, 171.5, 166.8, 158.1, 130.0, 125.7,115.1, 66.4, 54.1, 51.9, 51.4, 51.3, 42.6, 36.3, 31.1, 27.3, 24.1, 24.0,23.8, 22.0, 21.6.

HRMS (ES) 456.2444 (MNa⁺). C₂₃H₃₅N₃O₅ requires 456.2474.

FTIR (KBr) 3327, 2931, 862, 1743, 1681, 1654, 1541, 1508.

Microanalysis: C, 58.68; H, 7.62; N, 8.63. C₂₃H₃₅N₃O₅ requires C, 58.69;H, 7.71; N, 8.93.

(8S,11S,14S)-14-Benzyloxycarbonylamino-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo[14.2.2]icosa-1(19),16(20),17-triene-8-carboxylicacid methyl ester (26)

Amine 25 (0.550 g, 1.17 mmol) was dissolved in anhydrous DMF (20 mL).Benzyl chloroformate (0.250 mL, 1.76 mmol) and DIPEA (0.815 mL, 4.68mmol) were added and the reaction mixture was stirred at rt for 18 hbefore being partitioned between EtOAc and 1M hydrochloric acid. Theaqueous phase was extracted twice more with EtOAc and the combinedorganic extracts were dried (MgSO₄), filtered and concentrated in vacuo.The crude material was purified by flash chromatography on silica usinga gradient of EtOAc/DCM to yield an off-white solid, 0.460 g, 69%.R_(f)=0.32 (30% EtOAc/DCM). m.p. 91-93° C.

¹H-NMR (500 MHz in (CD₃)₂SO) 8.09 (1H, d J=7.5 Hz, NH Leu), 7.42 (1H, dJ=6.9 Hz, NH Tyr), 7.30-7.36 (6H, m, NH Gly and Ar—H (CBZ)), 7.02 (2H, dJ=8.3 Hz, Ar—H Tyr), 6.71 (2H, d J=8.3 Hz, Ar—H Tyr), 5.06 (1H, d J=12.7Hz, OCH₂Ph), 5.01 (1H, d J=12.7 Hz, OCH₂Ph), 4.32-4.38 (1H, m, CHCH₂Ph),4.06-4.15 (3H, m, CHCO₂CH₃ and CHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂CH₂), 3.98(1H, td J=5.1 Hz, J=10.0 Hz, OCH₂CH₂CH₂CH₂CH₂), 3.57 (3H, s, CHCO₂CH₃),2.86 (1H, dd J=5.2 Hz, J=12.1 Hz, CHCH₂Ph), 2.68 (1H, dd J=12.1 Hz,J=12.1 Hz, CHCH₂Ph), 1.61-1.68 (2H, m, OCH₂CH₂CH₂CH₂CH₂), 1.54-1.60 (1H,m, OCH₂CH₂CH₂CH₂CH₂), 1.40-1.52 (3H, m, CHCH₂CH(CH₃)₂ andCHCH₂CH(CH₃)₂), 1.16-1.36 (5H, m, OCH₂CH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂CH₂and OCH₂CH₂CH₂CH₂CH₂), 0.82 (3H, d J=6.5 Hz, CHCH₂CH(CH₃)₂), 0.80 (3H, dJ=6.5 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 568.3051 (MH⁺). C₃₁H₄₁N₃O₇ requires 568.3023.

(8S,11S,14S)-8-Hydroxymethyl-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo-[14.2.2]icosa-1(19),16(20),17-trien-14-yl)-carbamicacid benzyl ester (27)

Methyl ester 26 (0.300 g, 0.528 mmol) was dissolved in anhydrous THF (30mL) under an atmosphere of argon. The resultant solution was cooled inice and 1M LiAlH₄ in diethyl ether was added (0.581 mL, 0.581 mmol) andstirring was continued in ice for a further 1 h and then at rt for 18 h.MeOH (20 mL) was added and stirred was continued for a further 10 minsbefore the reaction mixture was concentrated in vacuo. The residue waspartitioned between EtOAc and 1M aqueous KHSO₄. The aqueous phase wasextracted twice more EtOAc and the combined organic extracts were washedwith brine, dried (MgSO₄), filtered and concentrated in vacuo to yield abrown solid, 0.206 g, 72%. m.p. 208-210° C.

¹H-NMR (500 MHz in CD₃OD) 7.57 (1H, d J=9.1 Hz, NH Gly), 7.35 (1H, dJ=7.3 Hz, NH Leu), 7.23-7.32 (5H, m, Ar—H (CBZ)), 7.20 (1H, d J=6.7 Hz,NH Tyr), 6.97 (2H, d J=8.2 Hz, Ar—H Tyr), 6.67 (2H, d J=8.2 Hz, Ar—HTyr), 5.08 (1H, d J=12.2 Hz, OCH₂Ph), 5.02 (1H, d J=12.6 Hz, OCH₂Ph),4.21 (1H, dd J=6.7 Hz, J=12.0 Hz, CHCH₂Ph), 4.03-4.10 (2H, m,OCH₂CH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂), 3.90-3.97 (1H, m,OCH₂CH₂CH₂CH₂CH₂), 3.56-3.58 (1H, m, CHCH₂OH), 3.28 (2H, d J=5.4 Hz,CHCH₂OH), 2.87 (1H, dd J=5.0 Hz, J=12.0 Hz, CHCH₂Ph), 2.68 (1H, ddJ=12.0 Hz, J=12.0 Hz, CHCH₂Ph), 1.57-1.70 (2H, m, OCH₂CH₂CH₂CH₂CH₂),1.32-1.47 (4H, m, OCH₂CH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂),1.14 (5H, m, OCH₂CH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂CH₂ andOCH₂CH₂CH₂CH₂CH₂), 0.76 (6H, d J=6.6 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 540.3061 (MH⁺). C₃₀H₄₁N₃O₆ requires 540.3073.

Microanalysis: C, 65.76; H, 7.33; N, 7.29. C₃₀H₄₁N₃O₆.½H₂O requires C,65.67; H, 7.72; N, 7.66.

(8S,11S,14S)-8-Formyl-11-isobutyl-10,13-dioxo-2-oxa-9,12-diaza-bicyclo[14.2.2]icosa-1(19),16(20),17-trien-14-yl)-carbamicacid benzyl ester (28)

Alcohol 27 (0.200 g, 0.369 mmol) was dissolved in DMSO (5 mL) under anatmosphere of argon. The resultant solution was cooled in ice before DCM(5 mL), DIPEA (0.257 mL, 1.48 mmol) and sulfur trioxide.pyridine complex(0.235 g, 1.48 mmol) pre-dissolved in DMSO (5 mL) were added. Thereaction mixture was stirred at 0° C. for 2 h before being partitionedbetween EtOAc and 1M hydrochloric acid. The aqueous phase was extractedagain with EtOAc and the combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo to afford an off white solid, 0.269g, 90%. m.p. 112-113° C.

¹H-NMR (500 MHz in CDCl₃) 9.49 (1H, s, CHO), 7.31-7.39 (5H, m, Ar—H(CBZ)), 7.07 (2H, d J=7.9 Hz, Ar—H (Tyr)), 6.76 (2H, d J=7.9 Hz, Ar—H(Tyr)), 6.23 (1H, d J=7.5 Hz, NH Leu), 6.09 (1H, d J=6.8 Hz, NH Gly),5.64 (1H, d J=8.8 Hz, NH Tyr), 5.12 (2H, s, OCH₂Ph), 4.39 (1H, ddd J=5.4Hz, J=6.8 Hz, J=7.4 Hz, CHCHO), 4.26-4.36 (1H, m, CHCH₂Ph), 4.07-4.22(1H, m, OCH₂CH₂CH₂CH₂CH₂), 4.03 (1H, ddd J=5.1 Hz, J=7.5 Hz, J=10.2 Hz,CHCH₂CH(CH₃)₂), 3.09 (1H, dd J=4.9 Hz, J=12.6, CHCH₂Ph), 2.77 (1H, ddJ=11.2 Hz, J=12.6 Hz, CHCH₂Ph), 1.70-1.82 (3H, m, OCH₂CH₂CH₂CH₂CH₂ andCHCH₂CH(CH₃)₂), 1.39-1.62 (3H, m, OCH₂CH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂),1.14 (5H, m, OCH₂CH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂CH₂ andOCH₂CH₂CH₂CH₂CH₂), 0.88 (3H, d J=5.4 Hz, CHCH₂CH(CH₃)₂), 0.86 (3H, dJ=5.4 Hz, CHCH₂CH(CH₃)₂).

HRMS (ES) 538.2918 (MH⁺). C₃₀H₃₉N₃O₆ requires 538.2917.

Microanalysis: C, 64.10; H, 7.22; N, 7.17. C₃₀H₃₉N₃O₆1½H₂O requires C,63.81; H, 7.22; N, 7.44.

Reagents and Conditions: (i) Et₃N, allyl bromide, DCM, (55%); (ii)HCl_((g)), Et₂O, (100%); (iii) HATU, DIPEA, 43, DMF, (97%); (iv) 3×10mol % GSGC, 1,1,2-TCE, microwave, (72%); (v) H₂, 20 mol % Pd/C, MeOH,EtOAc (44%) (vi) 4M HCl, 1,4-dioxane, (100%); (vii) benzylchloroformate, DIPEA, DMF, (18%); (viii) DIBAL-H, DCM, (17%).

(R)-3-Allylsulfanyl-2-tert-butoxycarbonylamino-propionic acid methylester (29)

N—BOC-cysteine methyl ester (obtained from Sigma-Aldrich, Auckland, NewZealand) (5.00 g, 21.2 mmol) was dissolved in anhydrous DCM (30 mL)under an atmosphere of argon. Triethylamine (3.26 mL, 23.3 mmol) andallyl bromide (2.02 mL, 23.3 mmol) were added. The reaction mixture wasstirred at rt for eighteen h before being concentrated in vacuo. Theresidue was partitioned between EtOAc and 1M hydrochloric acid. Theorganic phase was washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo. The crude material was purified by flashchromatography on silica using a gradient of EtOAc and (50/70) petroleumether to yield a white solid, 3.22 g, 55%. R_(f)=0.19 (1/5(EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 5.65 (1H, tdd J=7.1 Hz, J=10.1 Hz, J=17.1 Hz,CH₂CHCH₂), 5.34 (1H, d J=8.1 Hz, NH), 5.03-5.10 (2H, m, CH₂CHCH₂),4.41-4.43 (1H, m, CHCO₂CH₃), 3.76 (3H, s, CHCO₂CH₃), 3.05-3.09 (2H, dJ=5.7 Hz, CH₂CHCH₂), 2.86 (1H, dd J=4.5 Hz, J=14.8 Hz, CHCH₂S), 2.77(1H, dd J=8.1 Hz, J=14.8 Hz, CHCH₂S), 1.41 (9H, s, C(CH₃)₃).

LRMS (ES) 276.1 (MH⁺). C₁₂H₂₁NO₄S requires 276.1.

(R)-3-Allylsulfanyl-2-amino-propionic acid methyl ester hydrochloride(30)

Methyl ester 29 (3.20 g, 11.6 mmol) was dissolved in 4M hydrogenchloride in 1,4-dioxane (40 mL). The resultant solution was stirred atrt for 18 h before being concentrated in vacuo to yield a white solid,2.46 g, 100%.

¹H-NMR (500 MHz in CD₃OD) 5.78 (1H, tdd J=7.2 Hz, J=10.0 Hz, J=17.1 Hz,CH₂CHCH₂), 5.13-5.21 (2H, m, CH₂CHCH₂), 4.23 (1H, dd J=4.4 Hz, J=8.1 Hz,CHCO₂CH₃), 3.75 (3H, s, CHCO₂CH₃), 3.17-3.21 (2H, d J=7.2 Hz, CH₂CHCH₂),3.07 (1H, dd J=4.4 Hz, J=14.8 Hz, CHCH₂S), 2.89 (1H, dd J=8.1 Hz, J=14.8Hz, CHCH₂S).

¹³C-NMR (75 MHz in CD₃OD) 177.5, 170.2, 133.4, 52.6, 52.0, 34.1, 30.0.

LRMS (ES) 176.0 (MH⁺). C₇H₁₃NO₂S requires 176.1.

(R)-2-{(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-3-methyl-butyrylamino}-3-allylsulfanyl-propionicacid methyl ester (31)

Carboxylic acid 43 (1.50 g, 3.57 mmol), HATU (2.71 g, 3.92 mmol) andamine 30 (0.831 g, 3.92 mmol) were dissolved in DMF (20 mL). DIPEA wasadded (2.49 mL, 14.3 mmol) and the reaction mixture was stirred at rtfor 18 h before being partitioned between EtOAc and 1M hydrochloricacid. The organic phase was washed sequentially with 1M hydrochloricacid and brine before being dried (MgSO₄), filtered and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a whitesolid, 2.00 g, 97%. R_(f)=0.34 (1/1 EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CD₃OD) 7.17 (2H, d J=8.4 Hz, Ar—H), 6.82 (2H, d J=8.4Hz, Ar—H), 6.05 (1H, tdd J=5.3 Hz, J=10.6 Hz, J=17.4 Hz, OCH₂CHCH₂),5.73-5.82 (1H, m, SCH₂CHCH₂), 5.06-5.41 (4H, m, OCH₂CHCH₂ andSCH₂CHCH₂), 4.54-4.61 (1H, m, CHCO₂CH₃), 4.49-4.53 (2H, m, OCH₂CHCH₂),4.22-4.36 (2H, m, CHCH₂Ph and CHCH(CH₃)₃), 3.76 (3H, s, CHCO₂CH₃),3.15-3.21 (2H, m, SCH₂CHCH₂), 2.74-3.04 (4H, m, CHCH₂Ph andCHCH₂SCH₂CHCH₂), 2.02-2.09 (1H, m, CHCH(CH₃)₃), 1.39 (9H, s, C(CH₃)₃),0.96 (3H, d J=6.7 Hz, CHCH(CH₃)₃), 0.94 (3H, d J=6.7 Hz, CHCH(CH₃)₃).

HRMS (ES) 578.2892 (MH⁺). C₂₉H₄₃N₃O₇S requires 578.2900.

(E/Z)-(9R,12S,15S)-15-tert-Butoxycarbonylamino-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),4,17(21),18-tetraene-9-carboxylicacid methyl ester (32)

Diene 31 (1.00 g, 1.73 mmol) was dissolved in 1,1,2-trichloroethane (100mL) under an atmosphere of argon. GSGC (0.147 g, 0.173 mmol) was addedand the reaction mixture was heated at reflux in the microwave (1200 W)for 20 mins. Two further additions of GSGC (0.147 g, 0.173 mmol) wereadded and after each the reaction mixture was subjected to a further 20mins heating in the microwave. The resultant solution was allowed tocool before being concentrated in vacuo. The crude material was purifiedby flash chromatography on silica using a gradient of EtOAc and (50/70)petroleum ether to yield a brown solid, 0.685 g, 72%. A 1:1.7 ratio ofgeometric isomers was obtained. R_(f)=0.25 and 0.23 (1/1 EtOAc/(50/70)petroleum ether).

¹H-NMR for major isomer from mixture (500 MHz in (CD₃)OD) 7.03 (2H, dJ=8.7 Hz, Ar—H), 6.81 (2H, d J=8.7 Hz, Ar—H), 5.67-5.81 (2H, m,OCH₂CHCHCH₂) and OCH₂CHCHCH₂), 4.62-4.70 (2H, m, OCH₂CH₂CH₂CH₂S), 4.58(1H, dd J=5.4 Hz, J=14.5 Hz, CHCO₂CH₃), 4.44-4.51 (1H, m, CHCH₂Ph),4.18-4.29 (1H, m, CHCH(CH₃)₂), 3.69 (3H, s, CHCO₂CH₃), 3.08-3.22 (2H, m,OCH₂CH₂CH₂CH₂S), 3.06-3.11 (1H, m, CHCH₂Ph), 2.86-2.97 (1H, m, CHCH₂Ph),2.67-2.86 (1H, m, CHCH₂S), 2.56-2.64 (1H, m, CHCH₂S), 1.81-1.89 (1H, m,CHCH(CH₃)₂), 1.42 (9H, s, C(CH₃)₃), 0.94 (3H, d J=6.9 Hz, CHCH(CH₃)₂),0.92 (3H, d J=6.9 Hz, CHCH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 7.11 (2H, d J=8.0 Hz,Ar—H), 6.75 (2H, d J=8.0 Hz, Ar—H), 0.85 (3H, d J=6.5 Hz, CHCH(CH₃)₂),0.80 (3H, d J=6.5 Hz, CHCH(CH₃)₂).

HRMS (ES) 550.2599 (MH⁺). C₂₇H₃₉N₃O₇S requires 550.2587.

(9R,12S,15S)-15-tert-Butoxycarbonylamino-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),17(21),18-triene-9-carboxylicacid methyl ester (33)

Olefin 32 (0.700 g, 1.27 mmol) was dissolved in a mixture of EtOAc (50mL) and MeOH (20 mL). 10% palladium on carbon catalyst was added (0.140g, 20%) and the reaction mixture was subjected to hydrogenation at rtand atmospheric pressure for 18 h. The mixture was filtered throughcelite and concentrated in vacuo to yield a brown solid, 0.309 g, 44%.

¹H-NMR (500 MHz in (CD₃)OD 7.03 (2H, d J=8.2 Hz, Ar—H), 6.76 (2H, dJ=8.2 Hz, Ar—H), 4.58 (1H, dd J=6.0 Hz, J=14.4 Hz, CHCO₂CH₃), 4.18-4.31(3H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂S), 3.98-4.02 (2H, m, OCH₂CH₂CH₂CH₂Sand CHCH(CH₃)₂), 3.84-3.89 (1H, m, OCH₂CH₂CH₂CH₂S), 3.70 (3H, s,CHCO₂CH₃), 2.84-3.03 (2H, m, CHCH₂Ph), 2.71-2.85 (2H, m, CHCH₂S),1.97-2.13 (1H, m, CHCH(CH₃)₂), 1.80-1.91 (2H, m, OCH₂CH₂CH₂CH₂S),1.73-1.79 (2H, m, OCH₂CH₂CH₂CH₂S), 1.42 (9H, s, C(CH₃)₃), 0.94 (3H, dJ=6.7 Hz, CHCH(CH₃)₂), 0.82 (3H, d J=6.7 Hz, CHCH(CH₃)₂).

HRMS (ES) 552.2739 (MH⁺). C₂₇H₄₁N₃O₇S requires 552.2743.

(9R,12S,15S)-15-Amino-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo-[15.2.2]-henicosa-1(20),17(21),18-triene-9-carboxylicacid methyl ester hydrochloride (34)

Methyl ester 33 (0.300 g, 0.544 mmol) was dissolved in 4M HCl in1,4-dioxane (20 mL). The resultant solution was stirred at rt for 18 hbefore being concentrated in vacuo to yield an off white solid, 0.265 g,100%.

¹H-NMR (500 MHz in (CD₃)₂SO) 8.66 (2H, bs, NH₂), 8.54 (1H, d J=7.3 Hz,NH), 8.18 (1H, d J=8.9 Hz, NH), 6.98 (2H, d J=8.3 Hz, Ar—H), 6.78 (2H, dJ=8.3 Hz, Ar—H), 4.38-4.42 (1H, m, CHCO₂CH₃), 4.02-4.28 (4H, m, CHCH₂Phand OCH₂CH₂CH₂CH₂S and OCH₂CH₂CH₂CH₂S), 3.89-3.95 (1H, m, CHCH(CH₃)₂),3.84-3.88 (1H, m, OCH₂CH₂CH₂CH₂S), 3.55 (3H, s, CHCO₂CH₃), 3.06-3.14(2H, m, CHCH₂Ph), 2.71-2.90 (2H, m, CHCH₂S), 1.93-1.98 (1H, m,CHCH(CH₃)₂), 1.60-1.75 (4H, m, OCH₂CH₂CH₂CH₂S and OCH₂CH₂CH₂CH₂S), 0.88(3H, d J=7.1 Hz, CHCH(CH₃)₂), 0.87 (3H, d J=7.1 Hz, CHCH(CH₃)₂).

LRMS (ES) 452.3 (MH⁺). C₂₂H₃₃N₃O₅ requires 452.2.

(9R,12S,15S)-15-Benzyloxycarbonylamino-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),17(21),18-triene-9-carboxylicacid methyl ester (35)

Amine 34 (0.200 g, 0.410 mmol) was dissolved in anhydrous DMF (10 mL).Benzyl chloroformate (0.0877 mL, 0.615 mmol) and DIPEA (0.286 mL, 1.64mmol) were added. The resultant reaction mixture was stirred at rt for18 h before being partitioned between EtOAc and 1M hydrochloric acid.The aqueous phase was extracted twice more with EtOAc and the combinedorganic extracts were dried (MgSO₄), filtered and concentrated in vacuo.The crude material was purified by flash chromatography on silica usinga gradient of EtOAc/DCM to yield an off-white solid, 0.0430 g, 18%.R_(f)=0.44 (30% EtOAc/DCM).

¹H-NMR (500 MHz in (CD₃OD)) (compound exists as a mixture of rotamers)7.17-7.50 (5H, m, Ar—H (CBZ)), 6.94-7.18 (2H, m, Ar—H (Tyr)), 6.62-6.84(2H, m, Ar—H (Tyr)), 5.02 (1H, d J=10.6 Hz, OCH₂Ph), 4.98 (1H, d J=10.6Hz, OCH₂Ph), 4.32-4.49 (1H, m, CHCO₂CH₃), 4.14-4.33 (5H, m, CHCH₂Ph andOCH₂CH₂CH₂CH₂S and OCH₂CH₂CH₂CH₂S), 3.80-3.94 (1H, m, CHCH(CH₃)₂), 3.70(3H, s, CHCO₂CH₃), 2.88-3.12 (2H, m, CHCH₂Ph), 2.65-2.90 (2H, m,CHCH₂S), 2.02-2.10 (1H, m, CHCH(CH₃)₂), 1.55-1.65 (4H, m, OCH₂CH₂CH₂CH₂Sand OCH₂CH₂CH₂CH₂S), 0.86-0.91 (6H, m, CHCH(CH₃)₂).

HRMS (ES) 586.2566 (MH⁺). C₃₀H₃₉N₃O₇S requires 586.2587.

(9R,12S,15S)-9-Formyl-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo-[15.2.2]henicosa-1(20),17(21),18-trien-15-yl)-carbamicacid benzyl ester (36)

Methyl ester 35 (0.0430 g, 0.0734 mmol) was dissolved in anhydrous DCMunder an atmosphere of argon. The resultant solution was cooled to −78°C. and a 1M solution of DIBAL-H in hexanes (0.403 mL, 0.404 mmol) wasadded dropwise. The mixture was stirred at −78° C. for three h andsuper-dry methanol (1:1 ratio with DCM initially added), precooled to−78° C., was added dropwise. Stirring was continued at −78° C. for afurther twenty five min before the cooling bath was removed and 1Mhydrochloric acid was added. The organic layer was separated from theresulting white precipitate, diluted with EtOAc and allowed topartition. The organic phase was washed sequentially with 1Mhydrochloric acid, saturated aqueous NaHCO₃ and brine before being dried(MgSO₄), filtered and concentrated in vacuo. The crude material waspurified by flash chromatography on silica using EtOAc to yield a brownsolid, 0.00660 g, 17%. R_(f)=0.51 (EtOAc).

¹H-NMR (500 MHz in (CD₃)₂SO) 9.39 (1H, s, CHO), 7.70 (1H, d J=9.1 Hz, NHCys), 7.66 (1H, d J=8.1 Hz, NH Val), 7.12-7.43 (5H, m, Ar—H (CBZ)), 7.05(2H, d J=7.8 Hz, Ar—H (Tyr)), 6.78 (2H, d J=7.8 Hz, Ar—H (Tyr)), 6.62(1H, d J=8.3 Hz, NH), 5.03 (2H, s, OCH₂Ph), 4.20-4.32 (3H, m, CHCO₂CH₃and OCH₂CH₂CH₂CH₂S), 4.05-4.15 (3H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂S), 3.85(1H, dd J=6.5 Hz, J=8.1 Hz, CHCH(CH₃)₂), 2.78-2.90 (2H, m, CHCH₂Ph),2.54-2.70 (2H, m, CHCH₂S), 1.94-2.00 (1H, m, CHCH(CH₃)₂), 1.21-1.71 (4H,m, OCH₂CH₂CH₂CH₂S and OCH₂CH₂CH₂CH₂S), 0.82-0.88 (6H, m, CHCH(CH₃)₂).

HRMS (ES) 586.2480 (MH⁺). C₂₉H₃₇N₃O₆S requires 556.2481.

Reagents and Conditions: (i) a) allyl alcohol, ethyl chloroformate,Et₃N, Et₂O b) allyl palladium chloride, PPh₃, THF (21%); (ii) 4M HCl,1,4-dioxane, (87%); (iii) HATU, DIPEA, 43, DMF, (62%); (iv) 3×10 mol %GSGC, 1,1,2-TCE, microwave, (23%); (v) H₂, 20 mol % Pd/C, MeOH, EtOAc(100%).

(S)-3-Allyloxy-2-tert-butoxycarbonylamino-propionic acid methyl ester(37)

Allyl alcohol (1.97 mL, 29.0 mmol) was dissolved in diethyl ether (20mL). This was cooled in ice before ethyl chloroformate (0.304 mL, 31.9mmol) and triethylamine (0.445 mL, 31.9 mmol) were added. After stirringin ice for 20 mins the resultant white precipitate was removed bysuction filtration. The filtrate was concentrated in vacuo and theresidue dissolved in THF (4 mL). To the resultant solution a solution ofallyl palladium chloride dimer (0.0849 g, 0.232 mmol) andtriphenylphosphine (0.266 g, 1.02 mmol) in THF (3 mL) was added. Themixture was stirred at rt for twenty min and to this was added asolution of N-Boc-serine methyl ester (obtained from GL Biochem(Shanghai) Ltd., Shanghai, China) (5.00 g, 2.32 mmol) in THF (20 mL).The reaction mixture was stirred at rt for a further 18 h before beingconcentrated in vacuo. The crude material was purified by flashchromatography on silica using a gradient of EtOAc and (50/70) petroleumether to yield a yellow oil, 1.25 g, 21%. R_(f)=0.27 (1/6 EtOAc/(50/70)petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 5.64 (1H, tdd J=5.5 Hz, J=5.5 Hz, J=11.0 Hz,J=16.0 Hz, OCH₂CHCH₂), 5.37 (1H, d J=8.6 Hz, NH), 4.97-5.12 (2H, m,OCH₂CHCH₂), 4.21-4.24 (1H, m, CHCO₂CH₃), 3.74-3.80 (2H, m, OCH₂CHCH₂),3.65 (1H, dd J=3.2 Hz, J=9.4 Hz, CHCH₂OCH₂CHCH₂), 3.55 (3H, s,CHCO₂CH₃), 3.46 (1H, dd J=3.4 Hz, J=9.4 Hz, CHCH₂OCH₂CHCH₂), 1.26 (9H,s, C(CH₃)₃).

¹³C-NMR (75 MHz in CD₃OD) 173.3, 171.4, 133.6, 118.6, 66.6, 53.1, 30.5,26.7.

LRMS (ES) 260.2 (MH⁺). C₁₂H₂₁NO₅ requires 260.1.

(S)-3-Allyloxy-2-amino-propionic acid methyl ester hydrochloride (38)

Methyl ester 37 (1.00 g, 3.86 mmol) was dissolved in 4M hydrogenchloride in 1,4-dioxane (40 mL). The resultant solution was stirred atrt for 18 h before being concentrated in vacuo to afford a white solid,0.656 g, 87%. m.p. 87-89° C.

¹H-NMR (500 MHz in CD₃OD) 5.84-5.92 (1H, m, OCH₂CHCH₂), 5.19-5.31 (2H,m, OCH₂CHCH₂), 4.27 (1H, dd J=3.2 Hz, J=4.6 Hz, CHCO₂CH₃), 3.99-4.09(2H, m, OCH₂CHCH₂), 3.85-3.91 (1H, m, CHCH₂OCH₂), 3.83 (3H, s,CHCO₂CH₃), 3.79 (1H, dd J=3.2 Hz, J=10.6 Hz, CHCH₂OCH₂).

LRMS (ES) 160.1 (MH⁺). C₇H₁₃NO₃ requires 160.1.

(S)-3-Allyloxy-2-{(S)-2-[(S)-3-(4-allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-3-methyl-butyrylamino}-propionicacid methyl ester (39)

Carboxylic acid 43 (1.29 g, 3.07 mmol), HATU (1.28 g, 3.38 mmol) andamine 38 (0.660 g, 3.38 mmol) were dissolved in DMF (20 mL). DIPEA wasadded (2.14 mL, 12.3 mmol) and the reaction mixture was stirred at rtfor 18 h before being partitioned between EtOAc and 1M hydrochloricacid. The organic phase was washed sequentially with 1M hydrochloricacid and brine before being dried (MgSO₄), filtered and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a whitesolid, 1.07 g, 62%. R_(f)=0.46 (1/1 EtOAc/(50/70) petroleum ether). m.p.109-111° C.

¹H-NMR (500 MHz in CD₃OD) 7.12 (2H, d J=8.0 Hz, Ar—H), 6.82 (2H, d J=8.0Hz, Ar—H), 6.04 (1H, tdd J=5.3 Hz, J=5.3 Hz, J=10.4 Hz, J=17.1 Hz,PhOCH₂CHCH₂), 5.87 (1H, dddd J=5.5 Hz, J=5.6 Hz, J=11.0 Hz, J=16.0 Hz,CH₂OCH₂CHCH₂), 5.13-5.40 (4H, m, PhOCH₂CHCH₂ and CH₂OCH₂CHCH₂), 4.59(1H, dd J=3.2 Hz, J=4.7 Hz, CHCO₂CH₃), 4.46-4.53 (2H, m, PhOCH₂CHCH₂),4.25-4.32 (2H, m, CHCH₂Ph and CHCH(CH₃)₂), 4.02 (1H, dd J=5.5 Hz, J=13.0Hz, CH₂OCH₂CHCH₂), 3.97 (1H, dd J=5.6 Hz, J=13.0 Hz, CH₂OCH₂CHCH₂), 3.83(1H, dd J=4.7 Hz, J=9.8 Hz, CH₂OCH₂CHCH₂), 3.72 (3H, s, CHCO₂CH₃), 3.67(1H, dd J=3.8 Hz, J=8.7 Hz, CH₂OCH₂CHCH₂), 102 (1H, dd J=5.5 Hz, J=13.9Hz, CHCH₂Ph), 2.75 (1H, dd J=8.9 Hz, J=13.9 Hz, CHCH₂Ph), 1.95-2.06 (1H,m, CHCH(CH₃)₂), 1.37 (9H, s, C(CH₃)₃), 0.98 (3H, d J=6.7 Hz,CHCH(CH₃)₂), 0.94 (3H, d J=6.7 Hz, CHCH(CH₃)₂).

HRMS (ES) 562.3121 (MH⁺). C₂₉H₄₃N₃O₈ requires 562.3128.

(E/Z)-(9S,12S,15S)-15-tert-Butoxycarbonylamino-12-isopropyl-11,14-dioxo-2,7-dioxa-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),4,17(21),18-tetraene-9-carboxylicacid methyl ester (40)

Diene 39 (1.06 g, 1.89 mmol) was dissolved in 1,1,2-trichloroethane (150mL) under an atmosphere of argon. To the resultant solution GSGC (0.160g, 0.189 mmol) was added. The reaction mixture was heated at reflux inthe microwave (1200 W) for 20 mins. Two further additions of GSGC (0.160g, 0.189 mmol) were added and after each the reaction mixture wassubjected to a further 20 mins heating in the microwave. After coolingthe reaction mixture was concentrated in vacuo and the crude materialwas purified by flash chromatography on silica using a gradient of EtOAcand (50/70) petroleum ether to yield a brown solid, 0.233 g, 23%. A1:4.7 ratio of geometric isomers was obtained. R_(f)=0.28 and 0.27 (1/1EtOAc/(50/70) petroleum ether).

¹H-NMR for major isomer from mixture (500 MHz in CDCl₃) 7.09 (2H, dJ=8.4 Hz, Ar—H), 6.77 (2H, d J=8.4 Hz, Ar—H), 6.27 (1H, d J=8.3 Hz, NHVal), 6.24 (1H, d J=8.3 Hz, NH Ser), 5.73 (1H, ddd J=6.5 Hz, J=6.7 Hz,J=7.5 Hz, PhOCH₂CHCHCH₂), 5.62 (1H, td J=6.1 Hz, J=6.1 Hz, J=7.5 Hz,PhOCH₂CHCHCH₂), 5.40 (1H, d J=8.6 Hz, NH Tyr), 4.76 (1H, ddd J=4.0 Hz,J=4.3 Hz, J=8.3 Hz, CHCO₂CH₃), 4.67-4.61 (2H, m, PhOCH₂CHCHCH₂), 4.36(1H, ddd J=4.2 Hz, J=8.6 Hz, J=9.6 Hz, CHCH₂Ph), 4.13 (1H, dd J=6.7 Hz,J=13.6 Hz, CH₂OCH₂CHCHCH₂), 4.09 (1H, dd J=6.5 Hz, J=13.6 Hz,CH₂OCH₂CHCHCH₂), 3.80 (1H, dd J=7.7 Hz, J=8.3 Hz, CHCH(CH₃)₂), 3.78 (3H,s, CHCO₂CH₃), 3.58 (1H, dd J=4.3 Hz, J=9.1 Hz, CH₂OCH₂CHCHCH₂), 3.51(1H, dd J=4.0 Hz, J=9.1 Hz, CH₂OCH₂CHCHCH₂), 3.07 (1H, dd J=9.6 Hz,J=13.7 Hz, CHCH₂Ph), 2.91 (1H, dd J=4.2 Hz, J=13.7 Hz, CHCH₂Ph),1.99-2.08 (1H, m, CHCH(CH₃)₂), 1.46 (9H, s, C(CH₃)₃), 0.88 (3H, d J=6.7Hz, CHCH(CH₃)₂), 0.88 (3H, d J=6.7 Hz, CHCH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 6.80 (2H, d J=8.5 Hz,Ar—H), 4.33-4.25 (1H, m, CHCH₂Ph), 3.01 (1H, dd J=4.3 Hz, J=12.9 Hz,CHCH₂Ph), 0.85 (3H, d J=6.8 Hz, CHCH(CH₃)₂), 0.82 (3H, d J=6.5 Hz,CHCH(CH₃)₂).

HRMS (ES) 534.2820 (MH⁺). C₂₇H₃₉N₃O₈ requires 534.2815.

(9S,12S,15S)-15-tert-Butoxycarbonylamino-12-isopropyl-11,14-dioxo-2,7-dioxa-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),17(21),18-triene-9-carboxylicacid methyl ester (41)

Olefin 40 (0.200 g, 0.375 mmol) was dissolved in a mixture of EtOAc (20mL) and MeOH (20 mL). 10% palladium on carbon catalyst was added (0.0400g, 20%) and the reaction mixture was subjected to hydrogenation at rtand atmospheric pressure for 18 h. The mixture was filtered throughcelite and concentrated in vacuo to yield a brown solid, 0.200 g, 100%.

¹H-NMR (500 MHz in CD₃OD) 7.05 (2H, d J=8.3 Hz, Ar—H), 6.75 (2H, d J=8.3Hz, Ar—H), 4.60 (1H, dd J=3.9 Hz, J=6.3 Hz, CHCO₂CH₃), 4.21-4.36 (2H, m,CHCH₂Ph and PhOCH₂CH₂CH₂CH₂), 4.02-4.17 (3H, m, CHCH(CH₃)₂ andPhOCH₂CH₂CH₂CH₂ and CH₂OCH₂CH₂CH₂CH₂), 3.86-3.93 (1H, m,CH₂OCH₂CH₂CH₂CH₂), 3.69 (3H, s, CHCO₂CH₃), 3.47-3.55 (2H, m,CH₂OCH₂CH₂CH₂CH₂), 2.80-2.91 (2H, m, CHCH₂Ph), 1.92-2.05 (1H, m,CHCH(CH₃)₂), 1.71-1.90 (1H, m, PhOCH₂CH₂CH₂CH₂), 1.55-1.71 (3H, m,PhOCH₂CH₂CH₂CH₂ and PhOCH₂CH₂CH₂CH₂), 1.44 (9H, s, C(CH₃)₃), 0.87 (3H, dJ=6.4 Hz, CHCH(CH₃)₂), 0.86 (3H, d J=6.4 Hz, CHCH(CH₃)₂).

HRMS (ES) 536.2979 (MH⁺). C₂₇H₄₁N₃O₈ requires 536.2972.

Reagents and Conditions: (i) HATU, DIPEA, Val-OMe, DMF, (69%); (ii)NaOH, THF, H₂O, MeOH; (97%); (iii) HATU, DIPEA, (s)-allyl-Gly-OMe, DMF,(83%); (iv) 3×10 mol % GSGC, 10 mol % chloro-dicyclohexyl borane,1,1,2-TCE, microwave, (12%); (v) H₂, 20 mol % Pd/C, MeOH, EtOAc, (100%);(vi) 4M HCl, 1,4-dioxane, (100%); (vii) benzyl chloroformate, DIPEA,DMF, (70%); (viii) LiAlH₄, THF, (90%); (ix) SO₃.Pyr, DIPEA, DMSO, DCM,(81%).

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-3-methyl-butyricacid methyl ester (42)

N—BOC—O-allyl-tyrosine (5.0 g, 16.3 mmol), HATU (17.9 mmol) and valinemethyl ester hydrochloride (32.6 mmol) were dissolved in anhydrous DMF(25 mL). DIPEA (22.7 mL, 65 mmol) was added and the reaction mixture wasstirred at rt for 18 h before being partitioned between EtOAc and 1Mhydrochloric acid. The organic phase was then washed sequentially with1M hydrochloric acid and brine before being dried (MgSO₄), filtered andconcentrated in vacuo. Purification was achieved using flashchromatography, eluting with a gradient of (50/70) petroleum ether toEtOAc to yield a white solid, 6.08 g, 69%. R_(f)=0.29 (1/2 EtOAc/(50/70)petroleum ether). m.p. 74-76° C.

¹H-NMR (500 MHz in CDCl₃) 7.09 (2H, d J=6.5 Hz, Ar—H), 6.80 (2H, d J=6.5Hz, Ar—H), 6.47 (1H, bs, NH Val), 5.97-6.05 (1H, m, OCH₂CHCH₂),5.01-5.40 (2H, m, OCH₂CHCH₂), 5.12 (1H, bs, NH Tyr), 4.47-4.49 (2H, m,OCH₂CHCH₂), 4.37-4.46 (1H, m, CHCH₂Ph), 4.30-4.34 (1H, m, CHCH(CH₃)₂),3.66 (3H, s, CO₂CH₃), 3.01-3.04 (2H, m, CHCH₂Ph), 2.04-2.11 (1H, m,CHCH(CH₃)₂), 1.39 (9H, s, C(CH₃)₃), 0.85 (3H, d J=6.8 Hz, CHCH(CH₃)₂),0.82 (3H, d J=6.8 Hz, CHCH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 174.7, 171.9, 157.5, 133.2, 130.3, 128.6,117.6, 114.8, 68.7, 57.2, 37.0, 31.0, 28.2, 18.8, 17.6.

HRMS (ES) 435.2501 (MH⁺). C₂₃H₃₄N₂O₆ requires 435.2495.

Microanalysis: C, 62.58; H, 7.66; N, 6.58. C₂₃H₃₄N₂O₆ requires C, 62.84;H, 7.67; N, 6.66.

(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylamino-propionylamino]-3-methyl-butyricacid (43)

Methyl ester 42 (4.90 g, 11.3 mmol) was dissolved in THF (35 mL) andsodium hydroxide (17 mmol) pre-dissolved in water (10 mL) was added. Afurther 15 mL of THF and 20 mL of methanol were added to obtain ahomogenous solution. The reaction mixture was stirred at rt for 18 hbefore being concentrated in vacuo. The residue was partitioned betweenEtOAc and 1M hydrochloric acid. The aqueous phase was extracted twicemore with EtOAc and the combined organic extracts were washed withbrine, dried (MgSO₄), filtered and concentrated in vacuo to yield awhite foam, 4.60 g, 97%.

¹H-NMR (500 MHz in CDCl₃) 7.11 (2H, d J=8.3 Hz, Ar—H), 6.83 (2H, d J=8.3Hz, Ar—H), 6.64 (1H, d J=8.2 Hz, NH Val), 5.98-6.07 (1H, m, OCH₂CHCH₂),5.00-5.39 (2H, m, OCH₂CHCH₂), 5.19 (1H, bs, NH Tyr), 4.51 (3H, m,CHCH₂Ph and OCH₂CHCH₂), 4.36-4.40 (1H, m, CHCH(CH₃)₂), 2.97-3.02 (2H, m,CHCH₂Ph), 2.16-2.23 (1H, m, CHCH(CH₃)₂), 1.40 (9H, s, C(CH₃)₃),0.88-0.93 (6H, m, CHCH(CH₃)₂).

LRMS (ES) 421.3 (MH⁺). C₂₂H₃₂N₂O₆ requires 421.2.

(S)-2-{(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-tert-butoxycarbonylaminopropionylamino]-3-methylbutyrylamino}-pent-4-enoic acid methyl ester(44)

Carboxylic acid 43 (1.00 g, 2.38 mmol), HATU (2.62 mmol) and(S)-allyl-glycine methyl ester hydrochloride (2.62 mmol) were dissolvedin DMF (30 mL). DIPEA was added (9.52 mmol) and the reaction mixture wasstirred at rt for 18 h before being partitioned between EtOAc and 1Mhydrochloric acid. The organic phase was washed sequentially with 1Mhydrochloric acid and brine before being dried (MgSO₄), filtered andconcentrated in vacuo. Purification was achieved using flashchromatography, eluting with a gradient of EtOAc and (50/70) petroleumether to yield a white solid, 1.05 g, 83%. R_(f)=0.48 (1/1 EtOAc/(50/70)petroleum ether). m.p. 106-108° C.

¹H-NMR (500 MHz in CDCl₃) 7.09 (2H, d J=8.6 Hz, Ar—H), 6.82 (2H, d J=8.6Hz, Ar—H), 6.61 (1H, d J=8.4 Hz, NH Val), 6.56 (1H, d J=6.8 Hz, NH Gly),6.03 (1H, tdd J=5.3 Hz, J=10.6 Hz, J=17.2 Hz, OCH₂CHCH₂), 5.62-5.71 (1H,m, CHCH₂CHCH₂), 5.10-5.41 (4H, m, OCH₂CHCH₂ and CHCH₂CHCH₂), 5.05 (1H, dJ=5.5 Hz, NH Tyr), 4.58-4.62 (1H, m, CHCH₂CHCH₂), 4.48-4.50 (2H, m,OCH₂CHCH₂), 4.30-4.33 (1H, m, CHCH₂Ph), 4.25 (1H, dd J=6.5 Hz, J=8.4 Hz,CHCH(CH₃)₂), 3.73 (3H, s, CO₂CH₃), 2.96-3.05 (2H, m, CHCH₂Ph), 2.46-2.59(2H, m, CHCH₂CHCH₂), 2.07-2.14 (1H, m, CHCH(CH₃)₂), 1.39 (9H, s,C(CH₃)₃), 0.90 (3H, d J=6.8 Hz, CHCH(CH₃)₂), 0.86 (3H, d J=6.8 Hz,CHCH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 171.7, 171.4, 170.4, 157.6, 133.2, 132.2,130.0, 128.6, 119.3, 117.6, 114.9, 68.7, 58.4, 52.3, 51.7, 36.2, 30.7,28.2, 19.0.

HRMS (ES) 532.3010 (MH⁺). C₂₈H₄₂N₃O₇ requires 532.3023.

Microanalysis: C, 63.10; H, 7.76; N, 7.90. C₂₈H₄₂N₃O₇ requires C, 63.26;H, 7.77; N, 7.90.

(E)-(7S,10S,13S)-13-tert-Butoxycarbonylamino-10-isopropyl-9,12-dioxo-2-oxa-8,11-diazabicyclo[13.2.2]nonadeca-1(18),4,15(19),16-tetraene-7-carboxylicacid methyl ester (45)

Diene 44 (1.80 g, 3.39 mmol) was dissolved in anhydrous1,1,2-trichloroethane (0.01M) under an atmosphere of argon. GSGC (0.10equiv) was added. The mixture was heated at reflux for one hour. Twofurther additions of GSGC (0.10 equiv) were added and after each thereaction mixture was subjected to one h and eighteen h of refluxrespectively. This was then cooled and concentrated in vacuo. The crudematerial was purified by flash chromatography on silica using a gradientof EtOAc and (50/70) petroleum ether to yield a brown solid, 0.205 g,12%. R_(f)=0.24 (1/1 EtOAc/(50/70) petroleum ether). m.p. 105-107° C.

¹H-NMR (500 MHz in CDCl₃) 7.05 (2H, d J=8.6 Hz, Ar—H), 6.75 (2H, d J=8.6Hz, Ar—H), 5.74-5.75 (2H, m, NH Val and NH Gly), 5.43-5.56 (2H, m,OCH₂CHCHCH₂ and OCH₂CHCHCH₂), 5.32 (1H, d J=8.7 Hz, NH Tyr), 4.76 (1H,ddd J=3.4 Hz, J=9.2 Hz, J=10.1 Hz, CHCO₂CH₃), 4.58-4.64 (2H, m,OCH₂CHCHCH₂), 4.21 (1H, ddd J=5.2 Hz, J=8.7 Hz, J=11.6 Hz, CHCH₂Ph),3.99 (1H, dd J=4.8 Hz, J=7.5 Hz, CHCH(CH₃)₂), 3.75 (3H, s, CHCO₂CH₃),3.13 (1H, dd J=5.2 Hz, J=12.5 Hz, CHCH₂Ph), 2.66-2.75 (2H, m, CHCH₂Phand OCH₂CHCHCH₂), 2.26-2.32 (1H, m, OCH₂CHCHCH₂), 2.07-2.14 (1H, m,CHCH(CH₃)₂), 1.45 (9H, s, C(CH₃)₃), 0.81-0.83 (6H, m, CHCH(CH₃)₂).

HRMS (ES) 504.2718 (MH⁺). C₂₆H₃₇N₃O₇ requires 504.2710.

(7S,10S,13S)-13-tert-Butoxycarbonylamino-10-isopropyl-9,12-dioxo-2-oxa-8,11-diazabicyclo[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (46)

Olefin 45 (1.36 g, 2.70 mmol) was dissolved in 40 mL of EtOAc. 10%palladium on carbon catalyst was added (25%) and the reaction mixturewas subjected to hydrogenation at rt and atmospheric pressure for 18 hbefore being filtered through celite and concentrated in vacuo to yielda brown solid, 1.36 g, 100%. m.p. 225-228° C.

¹H-NMR (500 MHz in CDCl₃) 7.05 (2H, d J=8.0 Hz, Ar—H), 6.78 (2H, d J=8.0Hz, Ar—H), 6.23 (1H, d J=7.1 Hz, NH Val), 5.90 (1H, d J=8.2 Hz, NH Gly),5.29 (1H, d J=8.6 Hz, NH Tyr), 4.56 (1H, dt J=3.9 Hz, J=8.2 Hz,CHCO₂CH₃), 4.21-4.30 (2H, m, CHCH₂Ph, OCH₂CH₂CH₂CH₂) 4.09-4.13 (1H, m,OCH₂CH₂CH₂CH₂), 3.84-3.86 (1H, m, CHCH(CH₃)₂), 3.72 (3H, s, CHCO₂CH₃),3.10 (1H, dd J=5.4 Hz, J=12.2 Hz, CHCH₂Ph), 2.67 (1H, dd J=12.2 Hz,J=12.2 Hz, CHCH₂Ph), 1.95-2.02 (1H, m, CHCH(CH₃)₂), 1.86-1.92 (1H, m,OCH₂CH₂CH₂CH₂), 1.80 (1H, m, OCH₂CH₂CH₂CH₂), 1.49-1.57 (2H, m,OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂), 1.44 (9H, s, C(CH₃)₃), 1.25-1.35 (2H,m, OCH₂CH₂CH₂CH₂), 0.87 (3H, d J=6.6 Hz, CHCH(CH₃)₂), 0.81 (3H, d J=6.6Hz, CHCH(CH₃)₂).

¹³C-NMR (75 MHz in CDCl₃) 172.7, 170.3, 169.8, 156.9, 155.1, 130.0,128.4, 115.9, 79.6, 66.8, 57.6, 56.8, 52.3, 51.0, 38.4, 32.3, 31.7,28.2, 21.7, 18.2, 18.1.

HRMS (ES) 506.2871 (MH⁺). C₂₆H₄₀N₃O₇ requires 506.2866.

(7S,10S,13S)-13-Amino-10-isopropyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1-(18),15(19),16-triene-7-carboxylicacid methyl ester hydrogen chloride salt (47)

Methyl ester 46 (0.4 g) was dissolved in 4M HCl in 1,4-dioxane (10 mL).The solution was stirred at rt for 16 h before being concentrated invacuo to yield a brown solid, 0.38 g, 100%.

(7S,10S,13S)-13-Benzyloxycarbonylamino-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo-[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (48)

Amine 47 (65 mg) was dissolved in anhydrous DMF (30 mL). Benzylchloroformate (0.29 mL) and DIPEA (0.75 mL) were added and the reactionmixture was stirred at rt for 18 h before being partitioned betweenchloroform and 1M hydrochloric acid. The aqueous phase was extractedthree more times with chloroform and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 15%EtOAc/DCM to 50% EtOAc/DCM to yield an off-white solid, 54 mg, 70%.R_(f)=0.33 (30% EtOAc/DCM).

(7S,10S,13S)-7-Hydroxymethyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]-nona-deca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (49)

Methyl ester 48 (47 mg) was dissolved in anhydrous THF (40 mL) under anatmosphere of argon. The resultant solution was cooled in ice and 1MLiAlH₄ in diethyl ether was added (87 μL). The reaction mixture wasstirred in ice for 1 h and then at rt for 18 h. MeOH (10 mL) was addedand the reaction mixture was stirred at rt for a further 10 mins beforebeing concentrated in vacuo. The residue was partitioned between EtOAcand 1M aqueous KHSO₄. The aqueous phase was extracted twice more withchloroform and each organic extract was washed with brine before beingcombined, dried (MgSO₄), filtered and concentrated in vacuo to yield anoff-white solid, 40 mg, 90%.

(7S,10S,13S)-7-Formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (50)

Alcohol 49 (35 mg) was dissolved in DMSO (6, mL) under an atmosphere ofargon. To the resultant solution DCM (3 mL) and DIPEA (0.55 ml) wereadded. The reaction mixture was cooled in ice and sulfurtrioxide-pyridine complex (0.5 g) pre-dissolved in DMSO (15 mL) wasadded. This was stirred in ice for 2 h before being partitioned betweenEtOAc and 1M hydrochloric acid. The aqueous phase was extracted againwith EtOAc and the combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo. Purification was achieved usingflash chromatography, eluting with a gradient of 1/2 EtOAc/(50/70)petroleum ether to EtOAc to yield a light brown solid, 28 mg, 81%.R_(f)=0.61 (2/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) 1H-pyrrole-carboxylic acid, DIPEA, DMF,(55%); (ii) DIBAL, DCM, (33%).

(7S,10S,13S)-10-Isopropyl-9,12-dioxo-13-[(1H-pyrrole-2-carbonyl)-amino]-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (51)

Amine 47 (0.37 g) was dissolved in anhydrous DMF (5 mL).1H-pyrrole-carboxylic acid (0.11 g), DIPEA (0.72 mL), EDC (0.25 g) andHOBt (0.2 g) were added and the reaction mixture was stirred at rt for18 h before being partitioned between chloroform and 1M hydrochloricacid. The aqueous phase was extracted three more times with chloroformand the combined organic extracts were dried (MgSO₄), filtered andconcentrated in vacuo. Purification was achieved using flashchromatography, eluting with a gradient of 20% EtOAc/(50/70) petroleumether to EtOAc to yield an off-white solid, 279 mg, 55%. R_(f)=0.15 (20%EtOAc/(50/70) petroleum ether).

1H-Pyrrole-2-carboxylic acid((7S,10S,13S)-7-formyl-10-isopropyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-amide(52)

Ester 51 (45 mg) was dissolved in DCM (6 mL) under an atmosphere ofargon. The reaction was cooled to −78° C. To the resultant solutionDIBAL (0.5 mL) was added dropwise. This was stirred for 2 h before beingallowed to warm to rt overnight. The reaction mixture was partitionedbetween EtOAc and 1M hydrochloric acid. The aqueous phase was extractedagain with EtOAc and the combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo. Purification was achieved usingflash chromatography, eluting with a gradient of 1/1 EtOAc/(50/70)petroleum ether to EtOAc to yield a light brown solid, 14 mg, 33%.R_(f)=0.25 (1/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) 4F-benzene-sulfonyl chloride, DIPEA, DMF,(36%); (ii) DIBAL, DCM, (17%).

(7S,10S,13S)-13-(4-Fluoro-benzenesulfonylamino)-10-isopropyl-9,12-dioxo-2-oxa-8,1′-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (53)

Amine 47 (200 mg) was dissolved in anhydrous DMF (5 mL).4F-benzene-sulfonyl chloride (96 mg) and DIPEA (0.175 mL) were added andthe reaction mixture was stirred at rt for 18 h before being partitionedbetween chloroform and 1M hydrochloric acid. The aqueous phase wasextracted three more times with chloroform and the combined organicextracts were dried (MgSO₄), filtered and concentrated in vacuo.Purification was achieved using flash chromatography, eluting with agradient of 1/1 EtOAc/(50/70) petroleum ether to EtOAc to yield a lightbrown solid, 109 mg, 39%. R_(f)=0.15 (1/1 EtOAc/(50/70) petroleumether).

4-Fluoro-N-((7S,10S,135)-7-formyl-10-isopropyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-benzenesulfonamide(54)

Ester 53 (100 mg) was dissolved in DCM (6 mL) under an atmosphere ofargon. The reaction was cooled to −78° C. To the resultant solutionDIBAL (0.98 mL) was added dropwise. This was stirred for 2 h beforebeing allowed to warm to rt overnight. The reaction mixture waspartitioned between EtOAc and 1M hydrochloric acid. The aqueous phasewas extracted again with EtOAc and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 1/1EtOAc/(50/70) petroleum ether to EtOAc to yield a light brown solid, 16mg, 17%. R_(f)=0.35 (1/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) methane-sulfonyl chloride, DIPEA, DMF,(17%); (ii) DIBAL, DCM, (19%).

(7S,10S,13S)-10-Isopropyl-13-methanesulfonylamino-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-triene-7-carboxylicacid methyl ester (55)

Amine 47 (200 mg) was dissolved in anhydrous DMF (5 mL).Methane-sulfonyl chloride (39 μL) and DIPEA (0.175 mL) were added andthe reaction mixture was stirred at rt for 18 h before being partitionedbetween chloroform and 1M hydrochloric acid. The aqueous phase wasextracted three more times with chloroform and the combined organicextracts were dried (MgSO₄), filtered and concentrated in vacuo.Purification was achieved using flash chromatography, eluting with agradient of 1/1 EtOAc/(50/70) petroleum ether to EtOAc to yield a lightbrown solid, 40 mg, 17%. R_(f)=0.12 (1/1 EtOAc/(50/70) petroleum ether).

N-((7S,10S,13S)-7-Formyl-10-isopropyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-methanesulfonamide(56)

Ester 55 (40 mg) was dissolved in DCM (6 mL) under an atmosphere ofargon. The reaction was cooled to −78° C. To the resultant solutionDIBAL (0.46 mL) was added dropwise. This was stirred for 2 h beforebeing allowed to warm to rt overnight. The reaction mixture waspartitioned between EtOAc and 1M hydrochloric acid. The aqueous phasewas extracted again with EtOAc and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 1/1EtOAc/(50/70) petroleum ether to EtOAc to yield a light brown solid, 7mg, 19%. R_(f)=0.25 (1/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) 4F-benzene-sulfonyl chloride, DIPEA, DMF,(18%); (ii) DIBAL, DCM, (9%).

(9R,12S,15S)-15-(4-Fluoro-benzenesulfonylamino)-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),17(21),18-triene-9-carboxylicacid methyl ester (57)

Amine 34 (208 mg) was dissolved in anhydrous DMF (5 mL).4F-benzene-sulfonyl chloride (75 mg) and DIPEA (0.16 mL) were added andthe reaction mixture was stirred at rt for 18 h before being partitionedbetween chloroform and 1M hydrochloric acid. The aqueous phase wasextracted three more times with chloroform and the combined organicextracts were dried (MgSO₄), filtered and concentrated in vacuo.Purification was achieved using flash chromatography, eluting with agradient of 1/1 EtOAc/(50/70) petroleum ether to EtOAc to yield a lightbrown solid, 50 mg, 18%. R_(f)=0.2 (1/1 EtOAc/(50/70) petroleum ether).

4-Fluoro-N-((9R,12S,15S)-9-formyl-12-isopropyl-11,14-dioxo-2-oxa-7-thia-10,13-diaza-bicyclo[15.2.2]henicosa-1(20),17(21),18-trien-15-yl)-benzenesulfonamide(58)

Ester 57 (47 mg) was dissolved in DCM (6 mL) under an atmosphere ofargon. The reaction was cooled to −78° C. To the resultant solutionDIBAL (0.42°mL) was added dropwise. This was stirred for 2 h beforebeing allowed to warm to rt overnight. The reaction mixture waspartitioned between EtOAc and 1M hydrochloric acid. The aqueous phasewas extracted again with EtOAc and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 1/1EtOAc/(50/70) petroleum ether to EtOAc to yield a light brown solid, 9mg, 20%. R_(f)=0.45 (1/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) benzyl chloroformate, NaHCO₃, EtOAc, H₂O,(98%); (ii) NaIO₄, H₂O, MeOH, (100%); (iii) thermal elimination(kugelrohr), (20%); (iv) 6M HCl_((aq)), (71%); (v) SOCl₂, MeOH, (100%).

(S)-2-Benzyloxycarbonylamino-4-methylsulfanyl-butyric acid methyl ester(59)

L-methionine (3.70 g, 18.5 mmol) was dissolved in a biphasic mixture ofEtOAc (75 mL) and water (75 mL). This was cooled in ice and sodiumhydrogen bicarbonate (7.78 g, 5 equiv) and benzyl chloroformate (2.98mL, 1.1 equiv) were added. This was stirred in ice for one h and then atrt for a further eighteen h before the mixture was allowed to partition.The organic phase was washed sequentially with 1M hydrochloric acid,saturated aqueous sodium hydrogen bicarbonate and brine before beingdried (MgSO₄), filtered and concentrated in vacuo to afford a yellowoil, 5.40 g, 98%.

¹H-NMR (500 MHz in CDCl₃) 7.29 (5H, m, Ar—H), 5.79 (1H, d J=7.9 Hz, NH),5.08 (2H, s, CH₂Ph), 4.43-4.47 (1H, m, CHCO₂CH₃), 3.70 (3H, s,CHCO₂CH₃), 2.49 (2H, dd J=7.4 Hz, J=7.4 Hz, CHCH₂CH₂S), 2.11 (1H, dddJ=7.2 Hz, J=12.8 Hz, J=14.4 Hz, CHCH₂CH₂S), 2.04 (3H, s, SCH₃), 1.93(1H, ddd J=7.1 Hz, J=7.1 Hz, J=14.4 Hz, CHCH₂CH₂S).

LRMS (ES) 298.1 (MH⁺). C₁₄H₁₉NO₄S requires 298.1.

(S)-2-Benzyloxycarbonylamino-4-methanesulfinyl-butyric acid methyl ester(60)

Sulfide 59 (5.40 g, 18.2 mmol) was dissolved in methanol (60 mL). Thiswas cooled in ice and a solution of sodium metaperiodate (4.08 g, 1.05equiv) in water (25 mL) was added dropwise over 10 mins. This wasstirred in ice for 1 h and then at rt for a further 2 h. The resultantwhite precipitate was removed by filtration under vacuum, the residuewashed with methanol and the filtrate concentrated in vacuo. Theresultant colourless oil was partitioned between DCM and water. Theaqueous phase was extracted twice more with DCM, methanol added to thecombined organic extracts to obtain an homogenous solution and this wasdried (MgSO₄), filtered and concentrated in vacuo to afford a yellowoil, 5.69 g, 100%. This was obtained as a 1:1 mixture ofdiastereoisomers.

¹H-NMR (500 MHz in CDCl₃) 7.30-7.35 (5H, m, Ar—H), 6.00-6.08 (1H, m,NH), 5.10 (2H, s, CH₂Ph), 4.44-4.50 (1H, m, CHCO₂CH₃), 3.75 (3H, s,CHCO₂CH₃), 2.67-2.77 (2H, m, CHCH₂CH₂S), 2.53-2.54 (3H, m, SCH₃),2.32-2.40 (1H, m, CHCH₂CH₂S), 2.09-2.19 (1H, m, CHCH₂CH₂S).

¹³C-NMR (75 MHz in CDCl₃) 172.6, 156.0, 136.2, 128.6, 128.4, 128.3,128.1, 128.0, 69.9, 53.5, 53.0, 52.4, 31.6, 29.8, 15.3.

LRMS (ES) 314.1 (MH⁺). C₁₄H₁₉NO₅S requires 314.1.

(S)-2-Benzyloxycarbonylamino-but-3-enoic acid methyl ester (61)

Sulfoxide 60 (5.69 g, 18.2 mmol) was placed in a round bottomed flask ina kugelrohr. This was directly connected to a low vacuum diaphragm pumpand heated/distilled at 140° C. The distillate was purified by flashchromatography on silica using a gradient of EtOAc and (50/70) petroleumether to yield a colourless oil, 0.905 g, 20%. R_(f)=0.24 (1/7EtOAc/(50/70) petroleum ether).

¹H-NMR (500 MHz in CDCl₃) 7.31-7.35 (5H, m, Ar—H), 5.86-5.93 (1H, m,CHCH₂), 5.61 (1H, d J=6.3 Hz, NH), 5.01-5.39 (2H, m, CHCH₂), 5.11 (2H,s, CH₂Ph), 4.91-4.96 (1H, m, CHCO₂CH₃), 3.74 (3H, s, CHCO₂CH₃).

¹³C-NMR (75 MHz in CDCl₃) 170.9, 155.5, 136.1, 132.2, 128.5, 128.2,128.1, 127.5, 126.9, 117.7, 67.1, 56.1, 52.7.

LRMS (ES) 250.1 (MH⁺). C₁₃H₁₆NO₄ requires 250.1.

(S)-2-Benzyloxycarbonylamino-but-3-enoic acid methyl ester (62)

Olefin 61 (0.905 g, 3.61 mmol) was suspended in 6M hydrochloric acid (20mL). This was heated at reflux for 2 h, cooled and partitioned with DCM.The aqueous phase was washed again with DCM before being concentrated invacuo to afford a white solid. This was recrystallised from acetone toafford a white solid, 0.350 g, 71%.

¹H-NMR (500 MHz in (CD₃)₂SO) 8.32 (2H, bs, NH₂), 6.02-6.11 (1H, m,CHCH₂), 5.50-5.75 (2H, m, CHCH₂), 4.68-4.73 (1H, m, CHCO₂H).

(S)-2-Amino-but-3-enoic acid methyl ester hydrochloride (63)

Carboxylic acid 62 (0.350 g, 2.56 mmol) was suspended in methanol (10mL). This was cooled in ice and 20% (v/v) thionyl chloride was addedportionwise. The solution was stirred in ice for 1 h and then at rt for18 h before being concentrated in vacuo to afford a white solid, 0.388g, (100%).

¹H-NMR (500 MHz in CDCl₃) 8.70 (2H, bs, NH₂), 6.06-6.13 (1H, m, CHCH₂),5.52-5.73 (2H, m, CHCH₂), 4.79-4.82 (1H, m, CHCO₂CH₃), 3.83 (3H, s,CHCO₂CH₃).

Reagents and Conditions: (i) HATU, DIPEA, (S)-2-amino-but-3-enoic acidmethyl ester hydrochloride (63), DMF, (76%); (ii) 3×10 mol % GSGC, 10mol % chloro-dicyclohexyl borane, 1,1,2-TCE, microwave, (32%); H₂, 20mol % Pd/C, MeOH, EtOAc, (52%); (iv) 4M HCl, 1,4-dioxane, (100%); (v)4F-benzyl sulfonyl chloride, DIPEA, DMF, (17%); (vi) DIBAL, THF, (40%).

(S)-2-((S)-2-{(S)-3-(4-Allyloxy-phenyl)-2-[(tert-butoxy-hydroxy-methyl)-amino]-propionylamino}-3-methyl-butyrylamino)-but-3-enoicacid methyl ester (64)

Acid 43 (1.03 g, 2.45 mmol), amine 63 (1.1 equiv) and HATU (1.1 equiv)were dissolved in anhydrous DMF (0.10-0.50M relative to acid). DIPEA wasadded (4 equiv) and the reaction mixture stirred at rt for 18 h. Thiswas partitioned between EtOAc and 1M hydrochloric acid. The organicphase was washed sequentially with 1M hydrochloric acid and brine beforebeing dried (MgSO₄), filtered and concentrated in vacuo. The crudematerial was purified by flash chromatography on silica using a gradientof EtOAc and (50/70) petroleum ether to yield a white solid, 0.964 g,76%. R_(f)=0.46 (1/1 EtOAc/(50/70) petroleum ether). m.p. 104-106° C.

¹H-NMR (500 MHz in CDCl₃) 7.08 (2H, d J=8.2 Hz, Ar—H), 6.82 (2H, d J=8.2Hz, Ar—H), 6.62 (1H, d J=7.5 Hz, NH), 6.53 (1H, d J=8.5 Hz, NH), 6.42(1H, d J=8.3 Hz, NH), 6.07 (1H, tdd J=5.3 Hz, J=10.6 Hz, J=17.2 Hz,OCH₂CHCH₂), 5.90-5.97 (1H, m, CHCHCHCH₂), 5.05-5.42 (4H, m, OCH₂CHCH₂and CHCHCH₂), 4.95-5.00 (1H, m, CHCO₂CH₃), 4.49-4.51 (2H, m, OCH₂CHCH₂),4.25-4.38 (2H, m, CHCH₂Ph and CHCH(CH₃)₂), 3.74 (3H, s, CHCO₂CH₃),3.01-3.05 (2H, m, CHCH₂Ph), 2.04-2.11 (1H, m, CHCH(CH₃)₂), 1.45 (9H, s,C(CH₃)₃), 0.89 (3H, d J=7.1 Hz, CHCH(CH₃)₂), 0.87 (3H, d J=7.1 Hz,CHCH(CH₃)₂).

HRMS (ES) 518.2878 (MH⁺). C₂₇H₃₉N₃O₇ requires 518.2866.

Microanalysis: C, 62.37; H, 7.64; N, 8.11. C₂₇H₃₉N₃O₇ requires C, 62.65;H, 7.59; N, 8.12.

(E/Z)-(6S,9S,12S)-12-tert-Butoxycarbonylamino-9-isopropyl-8,11-dioxo-2-oxa-7,10-diazabicyclo[12.2.2]octadeca-1(17),4,14(18),15-tetraene-6-carboxylicacid methyl ester (65)

The diene 64 (0.830 g, 1.60 mmol) was dissolved in anhydrous1,1,2-trichloroethane (0.01M) under an atmosphere of argon. GSGC (0.10equiv) was added. The mixture was heated at reflux in the microwave(1200 W) for 20 min. Two further additions of GSGC (0.10 equiv) wereadded and after each the reaction mixture was subjected to a further 20min heating in the microwave. This was then cooled and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a brownsolid, 0.251 g, 32%. A 1:3.8 ratio of geometric isomers was obtained.R_(f)=0.32 and 0.35 (2/1 EtOAc/(50/70) petroleum ether).

¹H-NMR for major isomer from mixture (500 MHz in CDCl₃) 6.95 (2H, dJ=8.1 Hz, Ar—H), 6.78 (2H, d J=8.1 Hz, Ar—H), 5.84 (1H, d J=8.0 Hz, NHTyr), 5.38-5.47 (2H, m, OCH₂CHCHCH and OCH₂CHCHCH), 4.91-4.94 (1H, m,CHCO₂CH₃), 4.61-4.73 (2H, m, OCH₂CHCHCH), 4.04-4.13 (2H, m, CHCH₂Ph andCHCH(CH₃)₂), 3.78 (3H, s, CHCO₂CH₃), 3.03 (1H, dd J=5.1 Hz, J=12.5 Hz,CHCH₂Ph), 2.68 (1H, dd J=12.5 Hz, J=12.5 Hz, CHCH₂Ph), 1.92-1.97 (1H, m,CHCH(CH₃)₂), 1.45 (9H, s, C(CH₃)₃), 0.81-0.85 (6H, m, CHCH(CH₃)₂).

Selected ¹H-NMR for minor isomer from mixture: 7.19 (2H, d J=8.3 Hz,Ar—H), 4.22-4.30 (2H, m, CHCH₂Ph and CHCH(CH₃)₂), 3.74 (3H, s, CHCO₂CH₃)

HRMS (ES) 490.2546 (MH⁺). C₂₅H₃₅N₃O₇ requires 490.2553.

(6S,9S,12S)-12-tert-Butoxycarbonylamino-9-isopropyl-8,11-dioxo-2-oxa-7,10-diaza-bicyclo-[12.2.2]octadeca-1(17),14(18),15-triene-6-carboxylicacid methyl ester (66)

The olefin 65 (0.251 g, 0.513 mmol) was dissolved in methanol and 20%(w/w) of 10% palladium on carbon catalyst was added. The mixture wassubjected to hydrogenation at rt and atmospheric pressure for 18 h. Themixture was filtered through celite and concentrated in vacuo to afforda brown solid, 0.131 g, 52%.

¹H-NMR (500 MHz in CDCl₃) 6.96 (2H, d J=8.2 Hz, Ar—H), 6.71 (2H, d J=8.2Hz, Ar—H), 6.15 (1H, d J=7.1 Hz, NH Val), 5.93 (1H, d J=8.2 Hz, NH Gly),5.28 (1H, d J=8.6 Hz, NH Tyr), 4.53-4.55 (1H, m, CHCO₂CH₃), 4.23-4.31(1H, m, OCH₂CH₂CH₂CH) 4.05-4.22 (2H, m, CHCH₂Ph and OCH₂CH₂CH₂CH),3.82-3.84 (1H, m, CHCH(CH₃)₂), 3.76 (3H, s, CHCO₂CH₃), 3.12 (1H, ddJ=5.2 Hz, J=12.5 Hz, CHCH₂Ph), 2.62 (1H, dd J=12.5 Hz, J=12.5 Hz,CHCH₂Ph), 2.02-2.08 (1H, m, CHCH(CH₃)₂), 1.86-1.92 (1H, m,OCH₂CH₂CH₂CH), 1.80 (1H, m, OCH₂CH₂CH₂CH), 1.49-1.57 (2H, m,OCH₂CH₂CH₂CH and OCH₂CH₂CH₂CH), 1.47 (9H, s, C(CH₃)₃), 0.89 (3H, d J=6.8Hz, CHCH(CH₃)₂), 0.86 (3H, d J=6.8 Hz, CHCH(CH₃)₂).

HRMS (ES) 492.2700 (MH⁺). C₂₅H₃₇N₃O₇ requires 492.2710.

(6S,9S,12S)-12-Amino-9-isopropyl-8,11-dioxo-2-oxa-7,10-diaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-triene-6-carboxylicacid methyl ester hydrogen chloride salt (67)

Methyl ester 66 (0.2 g) was dissolved in 4M HCl in 1,4-dioxane (10 mL).The solution was stirred at rt for 16 h before being concentrated invacuo to yield a brown solid, 0.17 g, 100%.

(6S,9S,12S)-12-(4-Fluoro-benzenesulfonylamino)-9-isopropyl-8,11-dioxo-2-oxa-7,10-diaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-triene-6-carboxylicacid methyl ester (68)

Amine 67 (170 mg) was dissolved in anhydrous DMF (5 mL).4F-benzene-sulfonyl chloride (85 mg) and DIPEA (0.15 mL) were added andthe reaction mixture was stirred at rt for 18 h before being partitionedbetween chloroform and 1M hydrochloric acid. The aqueous phase wasextracted three more times with chloroform and the combined organicextracts were dried (MgSO₄), filtered and concentrated in vacuo.Purification was achieved using flash chromatography, eluting with agradient of 1/1 EtOAc/(50/70) petroleum ether to EtOAc to yield a lightbrown solid, 40 mg, 17%. R_(f)=0.2 (1/1 EtOAc/(50/70) petroleum ether).

4-Fluoro-N-((6S,9S,12S)-6-formyl-9-isopropyl-8,11-dioxo-2-oxa-7,10-diaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl)-benzenesulfonamide(69)

Ester 68 (39 mg) was dissolved in DCM (6 mL) under an atmosphere ofargon. The reaction was cooled to −78° C. To the resultant solutionDIBAL (0.39 mL) was added dropwise. This was stirred for 2 h beforebeing allowed to warm to rt overnight. The reaction mixture waspartitioned between EtOAc and 1M hydrochloric acid. The aqueous phasewas extracted again with EtOAc and the combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. Purification wasachieved using flash chromatography, eluting with a gradient of 1/1EtOAc/(50/70) petroleum ether to EtOAc to yield a light brown solid, 15mg, 40%. R_(f)=0.25 (1/1 EtOAc/(50/70) petroleum ether).

Reagents and Conditions: (i) HATU, DIPEA, (S)-2-amino-but-3-enoic acidmethyl ester hydrochloride (63), DMF, (83%); (ii) 3×10 mol % GSGC, 10mol % chloro-dicyclohexyl borane, 1,1,2-TCE, microwave, (48%); (iii) H₂,20 mol % Pd/C, MeOH, EtOAc, (53%).

2S-{2S-[3S-(4-Allyloxyphenyl)-2-tert-butoxycarbonylaminopropionylamino]-4-methylpentanoylamino}-but-3-enoicacid methyl ester (70)

Acid 2 (5.11 g, 11.8 mmol), amine 63 (1.1 equiv) and HATU (1.1 equiv)were dissolved in anhydrous DMF (0.10-0.50M relative to acid). DIPEA wasadded (4 equiv) and the reaction mixture stirred at rt for 18 h. Thiswas partitioned between EtOAc and 1M hydrochloric acid. The organicphase was washed sequentially with 1M hydrochloric acid and brine beforebeing dried (MgSO₄), filtered and concentrated in vacuo. The crudematerial was purified by flash chromatography on silica using a gradientof EtOAc and (50/70) petroleum ether to yield a white solid, 5.18 g,83%. R_(f)=0.5 (1/1 EtOAc/(50/70) petroleum ether). m.p. 102-104° C.

¹H-NMR (500 MHz in CDCl₃) 7.08 (2H, d J=8.4 Hz, o-Tyr), 6.96 (1H, dJ=6.3 Hz, vinylGly NH), 6.81 (2H, d J=8.5 Hz, m-Tyr), 6.49 (2H, d J=7.0Hz, Leu NH), 6.03 (1H, ddd J=5.3, J=10.5, J=22.4 Hz, allyl CH), 5.88(1H, ddd J=5.5, J=10.3, J=17.0 Hz, vinyl CH), 5.42-5.22 (4H, m, allylCH₂ and vinyl CH₂), 5.11-5.02 (2H, m, vinylGly α-H, Tyr NH), 4.61-4.40(3H, m, Leu α-H and OCH₂CHCH₂), 4.32 (1H, m, Tyr α-H), 3.76 (3H, s,OCH₃), 2.97 (2H, m, Tyr CH₂), 1.66 (1H, m, Leu CH₂), 1.57 (1H, m, LeuCH), 1.48 (1H, m, Leu CH₂), 1.39 (9H, s C(CH₃)₃), 0.90 (6H, dd J=4.5,J=6.2 Hz, Leu CH₃).

¹³C NMR (75 MHz in CDCl₃) 171.7, 171.4, 170.5, 157.5, 155.7, 133.2,131.6, 130.2, 128.6, 117.9, 117.5, 114.7, 80.0, 68.8, 55.7, 55.6, 54.4,52.6, 51.6, 40.9, 37.1, 28.2, 24.5, 22.7, 22.1.

HRMS (ES) 532.3027 (MH⁺). C₂₈H₄₂N₃O₇ requires 532.3023.

(E/Z)-12S-tert-Butoxycarbonylamino-9S-isobutyl-8,11-dioxo-2-oxa-7,10-diazabicyclo[12.2.2]octadeca-1(17),4,14(18),15-tetraene-6S-carboxylicacid methyl ester (71)

The diene 70 (1.06 g, 2.0 mmol) was dissolved in anhydrous1,1,2-trichloroethane (0.01M) under an atmosphere of argon. GSGC (0.10equiv) was added. The mixture was heated at reflux in the microwave(1200 W) for 20 mins. Two further additions of GSGC (0.10 equiv) wereadded and after each the reaction mixture was subjected to a further 20mins heating in the microwave. This was then cooled and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a whitesolid, 488 mg, 48%. m.p. 237-241° C. A >19:1 ratio of geometric isomerswas obtained. [α]_(D)=+1.6 (c 1, CHCl₃)

¹H NMR (500 MHz in CDCl₃) 7.17 (1H, d J=8.2 Hz, Ar—H), 6.87 (2H, ddJ=5.3, J=13.9 Hz, Ar—H), 6.72 (1H, d J=7.3 Hz, NH), 6.60 (1H, dd J=2.4,J=8.4 Hz, Ar—H), 5.88 (1H, d J=8.7 Hz, Leu NH), 5.72 (1H, ddd J=4.1,J=7.9, J=15.4 Hz, OCH₂CHCHCH), 5.49 (1H, d J=8.8 Hz, Tyr NH), 5.42 (1H,dd J=8.7, J=15.3 Hz, OCH₂CHCHCH), 4.89 (1H, t J=8.0 Hz, Gly Ha), 4.67(2H, m, OCH₂CHCHCH), 4.40 (1H, dd J=8.0, J=15.0 Hz, Leu Ha), 4.15 (1H,m, Tyr Ha), 3.78 (3H, s, OCH₃), 3.03 (1H, dd J=4.7, J=12.6 Hz, CHCH₂Ph),2.69 (1H, t J=12.3 Hz, CHCH₂Ph), 1.57-1.39 (12H, m, CHCH₂CH(CH₃)₂,CHCH₂CH(CH₃)₂, C(CH₃)₃), 0.87 (6H, t J=5.7 Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in CDCl₃) 170.7, 170.1, 155.3, 155.1, 130.8, 130.0,129.5, 129.4, 128.8, 119.4, 115.9, 79.6, 67.0, 57.3, 53.9, 52.8, 50.9,42.8, 38.7, 28.3, 24.6, 22.7, 22.6.

HRMS (ES) 504.2727. C₂₆H₃₈N₃O₇ requires 504.2710.

12S-tert-Butoxycarbonylamino-9S-isobutyl-8,11-dioxo-2-oxa-7,10-diazabicyclo[12.2.2]octadeca-1(17),14(18),15-triene-6S-carboxylicacid methyl ester (72)

The olefin 71 (450 mg, 0.89 mmol) was dissolved in methanol and 20%(w/w) of 10% palladium on carbon catalyst was added. The mixture wassubjected to hydrogenation at rt and atmospheric pressure for 18 h. Themixture was filtered through celite and concentrated in vacuo to afforda brown solid, 241 mg, 53%. m.p. 229-231° C. [α]_(D)=+20.8 (c 1, CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.20 (1H, d J=7.4 Hz, m-Tyr), 6.95 (1H, ddJ=2.2, J=8.3 Hz, o-Tyr), 6.88 (1H, dd J=2.6, J=8.4 Hz, o-Tyr), 6.69 (1H,dd J=2.6, J=8.3 Hz, m-Tyr), 6.11 (1H, d J=6.6 Hz, Gly NH), 5.94 (1H, dJ=7.8 Hz, Leu NH), 5.32 (1H, d J=8.6 Hz, Tyr NH), 4.48 (1H, dd J=5.8,J=10.3 Hz, Gly α-H), 4.31 (1H, m, OCH₂CH₂CH₂CH), 4.23-4.03 (3H, m, Tyrα-H, OCH₂CH₂CH₂CH, Leu α-H), 3.76 (3H, s, OCH₃), 3.14 (1H, dd J=5.5,J=12.5 Hz, CHCH₂Ph), 2.62 (1H, t J=12.1 Hz, CHCH₂Ph), 2.15 (1H, m,OCH₂CH₂CH₂CH), 1.92 (1H, m, OCH₂CH₂CH₂CH), 1.68 (1H, m, OCH₂CH₂CH₂CH),1.53-1.31 (12H, m, CHCH₂CH(CH₃)₂, C(CH₃)₃, CHCH₂CH(CH₃)₂), 1.23 (1H, m,OCH₂CH₂CH₂CH), 0.87 (6H, d J=6.5 Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in CDCl₃) 171.8, 170.2, 169.8, 155.7, 154.3, 129.9,129.2, 128.7, 118.4, 115.0, 79.1, 65.4, 56.6, 52.2, 51.3, 51.1, 43.0,38.7, 28.0, 26.7, 24.2, 22.4, 22.3, 20.8.

HRMS (ES) 506.2880 (MH⁺). C₂₆H₄₀N₃O₇ requires 506.2866.

Reagents and Conditions: (i) HATU, DIPEA, 30, DMF, (83%); (ii) 3×10 mol% GSGC, 10 mol % chloro-dicyclohexyl borane, 1,1,2-TCE, microwave,(40%); (iii) H₂, 20 mol % Pd/C, MeOH, EtOAc, (100%).

2S-{2S-[3S-(4-Allyloxyphenyl)-2-tert-butoxycarbonylaminopropionylamino]-4-methylpentanoylamino}-3-allylsulfanylpropionicacid methyl ester (73)

Acid 2 (933 mg, 2.15 mmol), amine 30 (1.1 equiv) and HATU (1.1 equiv)were dissolved in anhydrous DMF (0.10-0.50M relative to acid). DIPEA wasadded (4 equiv) and the reaction mixture stirred at rt for 18 h. Thiswas partitioned between EtOAc and 1M hydrochloric acid. The organicphase was washed sequentially with 1M hydrochloric acid and brine beforebeing dried (MgSO₄), filtered and concentrated in vacuo. The crudematerial was purified by flash chromatography on silica using a gradientof EtOAc and (50/70) petroleum ether to yield a white solid, 1.05 g,83%. m.p. 125-126° C. [α]_(D)=−19.4 (c 1, CHCl₃).

¹H NMR (500 MHz in d₆-acetone) 7.77 (1H, d J=7.8 Hz, Cys NH), 7.53 (1H,d J=7.9 Hz, Leu NH), 7.29 (1H, d J=8.5 Hz, o-Tyr), 6.97 (1H, d J=8.6 Hz,m-Tyr), 6.18 (2H, m, OCH₂CHCH₂, Tyr NH), 5.88 (1H, tdd J=7.2, J=10.0,J=17.1 Hz, SCH₂CHCH₂), 5.52 (1H, dd J=1.8, J=17.3 Hz, OCH₂CHCH₂), 5.34(1H, dd J=1.6, J=10.6 Hz, OCH₂CHCH₂), 5.27 (1H, dd J=1.6, J=17.0 Hz,SCH₂CHCH₂), 5.20 (1H, dd J=1.7, J=10.0 Hz, SCH₂CHCH₂), 4.76 (1H, dtJ=5.6, J=7.4 Hz, Cys α-H), 4.69 (1H, m, Leu α-H), 4.65 (2H, d J=5.2 Hz,OCH₂CHCH₂), 4.46 (1H, dt J=5.2, J=8.6 Hz, Tyr α-H), 3.82 (3H, s, OCH₃),3.29 (2H, t J=7.5 Hz, SCH₂CHCH₂), 3.23 (1H, dd J=4.8, J=13.9 Hz,CHCH₂Ph), 3.05 (1H, dd J=5.5, J=13.9 Hz, CHCH₂S), 3.02-2.95 (1H, m,CHCH₂Ph), 2.92 (1H, dd J=7.3, J=13.9 Hz, CHCH₂S), 1.82 (1H, m,CHCH₂CH(CH₃)₂), 1.78-1.63 (2H, m, CHCH₂CH(CH₃)₂), 1.47 (9H, s, C(CH₃)₃),1.02 (6H, t J=6.6 Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in d₆-acetone) 172.1, 171.6, 171.1, 157.6, 155.6, 134.4,134.2, 130.6, 130.1, 117.2, 116.5, 114.6, 78.7, 68.5, 56.1, 52.3, 51.9,51.4, 41.6, 37.1, 34.6, 32.0, 27.9, 24.5, 22.8, 21.5.

HRMS (ES) 614.2850 (MNa⁺). C₃₀H₄₅N₃O₇SNa requires 614.2876.

(E/Z)-15S-tert-Butoxycarbonylamino-12S-isobutyl-11,14-dioxo-2-oxa-2-thia-10,13-diaza-bicyclo[15.2.2]heneicosa-1(20),4,17(21),18-tetraene-9R-carboxylicacid methyl ester (74)

The diene 73 (503 mg, 0.85 mmol) was dissolved in anhydrous1,1,2-trichloroethane (0.01M) under an atmosphere of argon. GSGC (0.10equiv) was added. The mixture was heated at reflux in the microwave(1200 W) for 20 mins. Two further additions of GSGC (0.10 equiv) wereadded and after each the reaction mixture was subjected to a further 20mins heating in the microwave. This was then cooled and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a whitesolid, 197 mg, 40%. m.p. 228-231° C. A >19:1 ratio of geometric isomerswas obtained. [α]_(D)=−16.1 (c 1, CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.10 (2H, d J=8.2 Hz, Ar—H), 6.80 (2H, d J=8.3Hz, Ar—H), 6.53 (1H, d J=7.8 Hz, Cys NH), 5.90 (1H, d J=7.1 Hz, Leu NH),5.66 (2H, s, OCH₂CHCHCH₂S and OCH₂CHCHCH₂S), 5.37 (1H, d J=7.9 Hz, TyrNH), 4.72 (1H, d J=14.6 Hz, OCH₂CHCHCH₂S), 4.60 (2H, m, OCH₂CHCHCH₂S andCys α-H), 4.31 (1H, m, Leu α-H), 4.26 (1H, m, Tyr α-H), 3.78 (3H, sOCH₃), 3.19 (1H, dd J=5.6, J=15.5 Hz, OCH₂CHCHCH₂S), 3.07 (1H, d J=12.6Hz, OCH₂CHCHCH₂S), 2.94 (2H, m, CHCH₂Ph), 2.74-2.50 (2H, m, CHCH₂S),1.53 (2H, m, CHCH₂CH(CH₃)₂ and CHCH₂CH(CH₃)₂), 1.45 (10H, s, C(CH₃)₃ andCHCH₂CH(CH₃)₂), 0.86 (3H, d J=6.0 Hz, CHCH₂CH(CH₃)₂), 0.84 (d, 3H, J=5.7Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in CDCl₃) 170.8, 170.7, 170.4, 156.4, 154.7, 129.8,129.4, 128.6, 128.2, 114.7, 79.2, 66.9, 55.3, 51.9, 51.1, 50.9, 40.5,37.4, 32.4, 30.8, 28.0, 23.9, 22.5, 21.6.

HRMS (ES) 564.2752 (MH⁺). C₂₈H₄₂N₃O₇S requires 564.2743.

15S-tert-Butoxycarbonylamino-12S-isobutyl-11,14-dioxo-2-oxa-2-thia-10,13-diaza-bicyclo[15.2.2]heneicosa-1(20),17(21),18-triene-9R-carboxylicacid methyl ester (75)

The olefin 74 (188 mg, 0.334 mmol) was dissolved in methanol and 20%(w/w) of 10% palladium on carbon catalyst was added. The mixture wassubjected to hydrogenation at rt and atmospheric pressure for 18 h. Themixture was filtered through celite and concentrated in vacuo to afforda white solid, 188 mg, 100%. m.p. 210-211° C. [α]_(D)=+29.5 (c 1,CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.10 (2H, d J=7.8 Hz, o-Tyr), 6.79 (2H, dJ=7.9 Hz, m-Tyr), 6.24 (1H, d J=6.6 Hz, Cys NH), 6.06 (1H, d J=7.2 Hz,Leu NH), 5.33 (1H, d J=7.0 Hz, Tyr NH), 4.58 (1H, m, Cys α-H), 4.32-4.13(3H, m, Leu α-H, Tyr α-H, OCH₂CH₂CH₂CH₂S), 4.05 (1H, m, OCH₂CH₂CH₂CH₂S),3.76 (3H, s, OCH₃), 3.01 (1H, dd J=3.8, J=12.5 Hz, CHCH₂Ph), 2.82 (2H,m, CHCH₂Ph, CHCH₂S), 2.67 (1H, dd J=3.8, J=11.7 Hz, CHCH₂S), 2.56 (2H,m, OCH₂CH₂CH₂CH₂S), 1.92-1.65 (4H, m, OCH₂CH₂CH₂CH₂S, OCH₂CH₂CH₂CH₂S),1.53 (3H, m, CHCH₂CH(CH₃)₂, CHCH₂CH(CH₃)₂), 1.45 (9H, s, C(CH₃)₃), 0.86(6H, dd J=4.7, J=10.4 Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in CDCl₃) 171.2, 170.7, 170.4, 157.3, 155.2, 130.1,128.3, 115.1, 79.9, 66.5, 56.8, 52.7, 52.5, 51.2, 42.5, 38.2, 34.0,31.6, 28.3, 26.6, 25.6, 24.4, 22.8, 22.4.

HRMS (ES) 566.2903 (MH⁺). C₂₈H₄₄N₃O₇S requires 566.2900.

Reagents and Conditions: (i) HATU, DIPEA, 38, DMF, (77%); (ii) 3×10 mol% GSGC, 10 mol % chloro-dicyclohexyl borane, 1,1,2-TCE, microwave,(33%); H₂, 20 mol % Pd/C, MeOH, EtOAc, (75%).

3S-Allyloxy-2-{2S-[3S-(4-allyloxyphenyl)-2-tert-butoxycarbonylaminopropionylamino]-4-methyl-pentanoylamino}propionicacid methyl ester (76)

Acid 2 (4.58 g, 10.5 mmol), amine 38 (1.1 equiv) and HATU (1.1 equiv)were dissolved in anhydrous DMF (0.10-0.50M relative to acid). DIPEA wasadded (4 equiv) and the reaction mixture stirred at rt for 18 h. Thiswas partitioned between EtOAc and 1M hydrochloric acid. The organicphase was washed sequentially with 1M hydrochloric acid and brine beforebeing dried (MgSO₄), filtered and concentrated in vacuo. The crudematerial was purified by flash chromatography on silica using a gradientof EtOAc and (50/70) petroleum ether to yield a white solid, 4.65 g,77%. m.p. 87-89° C. [α]_(D)=−3.0 (c 1, CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.10 (2H, d J=8.5 Hz, o-Tyr), 6.83 (2H, dJ=8.6 Hz, m-Tyr), 6.68 (1H, d J=7.9 Hz, Ser NH), 6.40 (1H, d J=7.3 Hz,Leu NH), 6.04 (1H, ddd J=5.3, J=10.5, J=22.5 Hz, Tyr OCH₂CHCH₂), 5.84(1H, ddd J=5.6, J=10.8, J=16.0 Hz, Ser OCH₂CHCH₂), 5.39 (1H, dd J=1.6,J=17.3 Hz, Tyr OCH₂CHCH₂), 5.30-5.16 (3H, m, Tyr OCH₂CHCH₂, SerOCH₂CHCH₂), 4.95 (1H, br s, Tyr NH), 4.67 (1H, m, Ser α-H), 4.50 (2H, dJ=5.3 Hz, Tyr OCH₂CHCH₂), 4.46 (1H, m, Leu α-H), 4.31 (1H, m, Tyr α-H),3.98 (2H, dq, J=5.6, J=13.0 Hz, Ser OCH₂CHCH₂), 3.87 (1H, dd J=3.3,J=9.6 Hz, CHCH₂Oallyl), 3.75 (3H, s, OCH₃), 3.62 (1H, dd J=3.3, J=9.6Hz, CHCH₂Oallyl), 3.00 (2H, d J=6.6 Hz, CHCH₂Ar), 1.60 (2H, m,CHCH₂CH(CH₃)₂, CHCH₂CH(CH₃)₂), 1.48 (1H, m, CHCH₂CH(CH₃)₂), 1.40 (9H, sC(CH₃)₃), 0.91 (6H, dd J=2.8, J=6.2 Hz, CHCH₂CH(CH₃)₂)

¹³C NMR (75 MHz in CDCl₃ 171.5, 171.2, 170.3, 157.5, 155.4, 133.9,133.2, 130.2, 128.6, 117.5, 114.7, 80.1, 72.1, 69.3, 68.7, 55.6, 52.5,51.5, 41.4, 37.1, 28.2, 24.4, 22.8, 22.0

HRMS (ES) 576.3275 (MH⁺); C₃₀H₄₆N₃O₈ requires 576.3285.

(E/Z)-15S-tert-Butoxycarbonylamino-12S-isobutyl-11,14-dioxo-2,7-dioxa-10,13-diaza-bicyclo[15.2.2]heneicosa-1(20),4,17(21),18-tetraene-9R-carboxylicacid methyl ester (77)

The diene 76 (719 mg, 1.25 mmol) was dissolved in anhydrous1,1,2-trichloroethane (0.01M) under an atmosphere of argon. GSGC (0.10equiv) was added. The mixture was heated at reflux in the microwave(1200 W) for 20 min. Two further additions of GSGC (0.10 equiv) wereadded and after each the reaction mixture was subjected to a further 20min heating in the microwave. This was then cooled and concentrated invacuo. The crude material was purified by flash chromatography on silicausing a gradient of EtOAc and (50/70) petroleum ether to yield a palebrown solid, 229 mg, 33%. m.p. 209-211° C. A>19:1 ratio of geometricisomers was obtained. [α]_(D)=−0.30 (c 1, CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.08 (2H, d J=8.4 Hz, Ar—H), 6.76 (2H, d J=8.5Hz, Ar—H), 6.42 (1H, d J=8.2 Hz, Ser NH), 6.23 (1H, d J=7.9 Hz, Leu NH),5.72 (1H, td J=3.9, J=3.9, J=16.0 Hz, PhOCH₂CHCHCH₂O), 5.68-5.60 (1H, tdJ=6.2, J=6.2, 16.0 Hz PhOCH₂CHCHCH₂O), 5.36 (1H, d J=8.0 Hz, Tyr NH),4.72 (1H, m, Ser α-H), 4.64 (2H, s, PhOCH₂CHCHCH₂O), 4.39-4.26 (2H, m,Leu α-H, Tyr α-H), 4.12 (1H, dd J=5.1, J=12.9 Hz, PhOCH₂CHCHCH₂O), 3.81(1H, dd J=7.1, J=12.7 Hz, PhOCH₂CHCHCH₂O), 3.78 (3H, s, OCH₃), 3.53 (1H,dd J=5.3, J=8.9 Hz, CHCH₂OCH₂), 3.47 (1H, dd J=4.0, J=9.1 Hz,CHCH₂OCH₂), 3.09 (1H, dd J=9.2, J=13.4 Hz, CHCH₂Ar), 2.86 (1H, dd J=3.7,J=13.6 Hz, CHCH₂Ar), 1.62-1.47 (3H, m, CHCH₂CH(CH₃)₂, CHCH₂CH(CH₃)₂),1.46 (9H, s, C(CH₃)₃), 0.88 (6H, t J=6.5 Hz, CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz, CDCl₃) 171.0, 170.7, 170.1, 156.5, 155.2, 130.9, 130.1,128.3 (2C), 115.1, 79.8, 70.7, 68.4, 67.0, 55.2, 52.6, 51.9, 51.4, 41.9,37.3, 28.3, 24.4, 22.7, 22.2.

HRMS (ES) 548.2977 (MH⁺). C₂₈H₄₂N₃O₈ requires 548.2972.

15S-tert-Butoxycarbonylamino-12S-isobutyl-11,14-dioxo-2,7-dioxa-10,13-diaza-bicyclo[15.2.2]heneicosa-1(20),17(21),18-triene-9R-carboxylicacid methyl ester (78)

The olefin 77 (100 mg, 0.18 mmol) was dissolved in methanol and 20%(w/w) of 10% palladium on carbon catalyst was added. The mixture wassubjected to hydrogenation at rt and atmospheric pressure for 18 h. Themixture was filtered through celite and concentrated in vacuo to afforda white solid, 75 mg, 75%. m.p. 181-183° C. [α]_(D)=−0.30 (c 1, CHCl₃).

¹H NMR (500 MHz in CDCl₃) 7.14 (2H, d J=8.0 Hz, o-Tyr), 6.78 (2H, dJ=8.3 Hz, m-Tyr), 6.23 (1H, d J=7.6 Hz, Ser NH), 5.91 (1H, d J=6.8 Hz,Leu NH), 5.31 (1H, d J=8.4 Hz, Tyr NH), 4.66 (1H, m, Ser α-H), 4.25 (1H,m, Tyr α-H), 4.17 (2H, m, Leu α-H, Tyr OCH₂CH₂), 4.04 (1H, td J=5.3,J=5.3, 10.7 Hz, Tyr OCH₂CH₂), 3.77 (3H, s, OCH₃), 3.61 (2H, s,CHCH₂OCH₂), 3.55 (1H, m, Tyr OCH₂CH₂CH₂CH₂O), 3.46 (1H, m, TyrOCH₂CH₂CH₂CH₂O), 3.02 (1H, dd J=4.4, J=13.0 Hz, CHCH₂Ar), 2.88 (1H, tJ=11.9 Hz, CHCH₂Ar), 1.90-1.61 (4H, m, Tyr OCH₂CH₂CH₂CH₂O,CHCH₂CH(CH₃)₂), 1.62-1.48 (3H, m, Tyr OCH₂CH₂CH₂CH₂O, CHCH₂CH(CH₃)₂),1.45 (9H, s, C(CH₃)₃), 0.87 (6H, s CHCH₂CH(CH₃)₂).

¹³C NMR (75 MHz in CDCl₃) 171.1, 170.4, 170.3, 156.8, 155.1, 130.1,128.3, 115.3, 79.7, 71.3, 70.0, 67.1, 56.2, 53.0, 52.5, 51.9, 42.4,38.1, 28.2, 25.0, 24.7, 24.4, 22.7, 22.3.

HRMS (ES) 550.3115 (MH⁺). C₂₈H₄N₃O₈ requires 550.3128.

Reagents and Conditions: (i) Et₃N, MSCl, 9, DCM; (ii) NaN₃, DMF (61% forsteps (i) and (ii)); (iii) NaHSO₃, KCN (81%); (iv) cyclopropylamide,NaBH(OAc)₃, (61%).

(7S-azidomethyl-10S-isobutyl-9,12-dixoxo-2-oxa-8,11-diazabicyclo-[13.2.2]nonadeca-1(18),15(19),16-trien-13S-yl)carbamicacid benzyl ester (79)

To the macrocyclic alcohol 8 (0.19 g, 0.36 mmol, 1.0 equiv) indichloromethane was added triethylamine (120 μL, 0.90 mmol, 2.5 equiv)and mesyl chloride (16 μL, 0.36 mmol, 1.0 equiv). The reaction mixturewas stirred at rt overnight, then the solvent removed in vacuo. Theresidue was dissolved in DMF and sodium azide (0.02 g, 0.36 mmol, 1.0equiv) added and the reaction mixture stirred at rt for 4 h after whichit was diluted with EtOAc. The organic phase was washed with brine,dried over MgSO₄ and solvent removed under reduced pressure to give thecrude material. Recrystallisation from EtOAc/petroleum ether affordedthe product as a white solid, 0.12 g, 61%. m.p. >250° C. [α]_(D)=−3.0 (c0.1, (CH₃)₂SO).

¹H NMR (500 MHz in (CD₃)₂SO) 7.55 (1H, d J=9.1 Hz, NH), 7.52 (1H, dJ=7.1 Hz, NH), 7.38-7.35 (5H, m, Ar—H (Cbz)), 7.01 (2H, d J=7.5 Hz, Ar—H(o-Tyr)), 6.93 (1H, d J=7.7 Hz, NH), 6.74 (2H, d J=8.1 Hz, Ar—H(m-Tyr)), 5.03 (2H, q_(AB), J=12.6 Hz CCH₂O), 4.59-4.57 (1H, m,CHCH₂Ph), 4.34-4.29 (2H, m, OCH₂CH₂CH₂CH₂ and CHCH₂N₃), 4.06-4.01 (2H,m, OCH₂CH₂CH₂CH₂ and CHCH₂CH(CH₃)₂), 3.18-3.14 (1H, m, CHCH₂Ph),3.08-3.03 (1H, m, CHCH₂Ph), 2.86 (1H, dd J=5.6, J=13.1 Hz, CHCH₂N₃),2.62-2.60 (1H, m, CHCH₂N₃), 1.77-1.68 (2H, m, CHCH₂CH(CH₃)₂), 1.52-1.44(2H, m, OCH₂CH₂CH₂CH₂), 1.33-1.14 (4H, m, OCH₂CH₂CH₂CH₂ andCHCH₂CH(CH₃)₂), 0.87-0.82 (1H, m, OCH₂CH₂CH₂CH₂), 0.80-0.77 (6H, m,CHCH₂CH(CH₃)₂), 0.64-0.59 (1H, m, OCH₂CH₂CH₂CH₂).

¹³C NMR (75 MHz in (CD₃)₂SO) 170.92, 169.90, 156.54, 155.80, 137.46,130.45, 128.72, 128.60, 128.03, 115.03, 66.77, 65.65, 64.47, 56.62,51.13, 49.60, 43.63, 37.38, 30.26, 28.28, 24.24, 23.27, 23.12, 22.35.

ν_(max) (KBr) 2955 (C(O)NH), 2864 (C(O)NH), 2783 (C(O)NH), 2406 (CH₂N₃),1701 (OC(O)).

[7-(R,S)-(cyanohydroxymethyl)-10S-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo-[13.2.2]nonadeca-1(18),15(19),16-trien-13S-yl]carbamicacid benzyl ester (80)

To a cooled solution (5° C.) of the macrocyclic aldehyde 9 (0.50 g, 0.96mmol, 1 equiv) in methanol was added a cooled (5° C.) aqueous solutionof sodium hydrogen sulfite (1.0 equiv). The solution was stirred for 16h at 4° C. then potassium cyanide (1.0 equiv) in EtOAc added. Thebiphasic reaction mixture was stirred for 4 h at rt. The organic layerwas separated and the aqueous phase was extracted (×2) with EtOAc. Theseparated organic phases were combined, washed with distilled water,dried over MgSO₄ and the solvent removed to yield the cyanohydrin 80(0.43 g, 81%) as a mixture of diastereoisomers (1:1).

¹H NMR (500 MHz in (CD₃)₂SO) 7.99 (1H, d J=8.7 Hz, NH), 7.90 (1H, dJ=9.5 Hz, NH), 7.52 (1H, d J=7.4 Hz, NH), 7.35 (10H, s, Ar—H (Cbz)),7.31 (1H, d J=5.5 Hz, NH), 7.07 (1H, d J=8.3 Hz, NH), 7.03-6.99 (4H, m,Ar—H (o-Tyr), 6.96 (1H, d J=8.1 Hz, NH), 6.76 (4H, d J=7.8 Hz, Ar—H(m-Tyr), 6.61 (1H, d J=6.9 Hz CHCHOH), 6.55 (1H, d J=5.8 Hz CHCHOH),5.08-4.99 (4H, m, CCH₂O), 4.37-4.29 (4H, m, CHCH₂Ph and OCH₂CH₂CH₂CH₂),4.13-4.10 (1H, m, CHCH₂CH(CH₃)₂), 4.08-4.01 (3H, m, CHCH₂CH(CH₃)₂ andOCH₂CH₂CH₂CH₂), 3.98-3.94 (2H, m, OCH₂CH₂CH₂CH₂), 3.89-3.85 (2H, m,CHCHOH), 2.85 (2H, dd J=5.2, J=13.1 Hz, CHCH₂Ph), 2.64-2.58 (2H, m,CHCH₂Ph), 1.76-1.67 (2H, m, OCH₂CH₂CH₂CH₂), 1.61-1.45 (4H, m,CHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂), 1.37-1.12 (10H, m, CHCH₂CH(CH₃)₂ andCHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂ and OCH₂CH₂CH₂CH₂OCH₂CH₂CH₂CH₂),0.84-0.75 (14H, m, CHCH₂CH(CH₃)₂ and OCH₂CH₂CH₂CH₂).

¹³C NMR (75 MHz in (CD₃)₂SO) 171.19, 170.91, 169.41, 156.13, 155.76,155.25, 137.10, 130.03, 128.21, 127.65, 127.58, 119.86, 119.33, 115.39,115.34, 115.30, 66.18, 66.14, 65.11, 65.06, 63.40, 62.85, 56.04, 55.93,50.54, 50.50, 49.95, 49.74, 43.46, 43.43, 43.34, 43.28, 37.06, 30.02,29.17, 28.13, 27.52, 27.18, 23.88, 23.63, 22.91, 22.77, 22.60, 21.68,21.53.

HRMS (ES) 551.2876 (MH⁺). C₃₀H₃₉N₄O₆ requires 551.2870;

ν_(max) (KBr) 3319 (CH(OH)), 3064 (C(O)NH), 2922 (C(O)NH), 1882(CH(OH)CN).

((7S,10S,13S)-7-Cyclopropylaminomethyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (81)

Cyclopropylamine (29 μL, 0.42 mmol, 1.1 equiv.) was added to a stirredsolution of the macrocyclic aldehyde 9 (200 mg, 0.38 mmol, 1.0 equiv.)in freshly distilled 1,2-dichloroethane under a nitrogen atmosphere andstirred for 30 mins, at which time NaBH(OAc)₃ (113 mg, 0.53 mmol, 1.4equiv.) was added portionwise. After stirring for a further 22 h, thecloudy reaction mixture was quenched with saturated aqueous NaHCO₃,extracted with dichloromethane, washed with brine, dried (MgSO₄), andconcentrated in vacuo. Flash chromatography on activated alumina(eluting with 1.5% to 2% methanol in DCM) gave 81 as a white solid, 147mg, 68%. R_(f) 0.29 (2% methanol in DCM, alumina). m.p. 208° C. (dec.).

¹H NMR (500 MHz in (CD₃)₂SO) 0.89-0.92 (m, 6H, CH₂CH(CH₃)₂), 1.20-1.29(m, 1H, CH₂CH₂CH₂O), 1.31-1.36 (m, 1H, CH₂CH₂O), 1.38-1.45 (m, 4H,CH₂CH(CH₃)₂ and CH₂CH₂CH₂O), (m, 3H, CH₂CH(CH₃)₂ and CH₂CH₂CH₂CH₂O),1.77-1.86 (m, 1H, NHcyclopropyl), 2.07-2.09 (m, 1H, CH₂NHcyclopropyl),2.50-2.51 (m, 1H, CH₂NHcyclopropyl), 2.71 (t J=12.19 Hz, 1H, CH₂C₆H₄O),2.96 (dd J=12.86, J=5.53 Hz, 1H, CH₂C₆H₄O), 3.80-3.87 (m, 1H,CHCH₂NHcyclopropyl), 4.00-4.02 (CHCH₂CH(CH₃)₂), 4.12-4.16 (m, 1H, CH₂O),4.37-4.44 (m, 2H, Cbz NHCH and CH₂CO), 5.13 (q, J=12.75 Hz, 1H, CbzCH₂), 6.85 (d J=8.30 Hz, 1H, CH₂CO), 6.99 (d J=8.30 Hz, 1H,CbzNHCHCOCH), 7.11 (d J=7.06 Hz, 1H, CH₂CH₂CO), 7.41-7.18 (m, 5H, CbzAr—H), 7.61 (d J=7.17 Hz, 1H, Cbz NH), 7.65 (d J=9.16 Hz, 1H, CONH).

¹³C NMR (75 MHz in (CD₃)₂SO) 17.5, 17.7, 18.7, 18.9, 21.5, 21.6, 22.2,22.4, 24.6, 30.8, 42.1, 49.5, 61.3, 66.6, 127.6, 127.8, 127.8, 128.0,128.0, 128.2, 128.5, 137.0, 147.4, 147.7, 157.4, 161.3, 172.4, 172.4.

HRMS 565.3375 [MH⁺]. C₃₂H₄₅N₄O₅ requires 565.3390.

Reagents and Conditions: (i) SO₂Cl, MeOH (99%); (ii) EDC.HCl, HOBt,DIPEA, DMF, (76%).

(S)-2-Amino-6-hydroxy-hexanoic acid methyl ester (82)

(S)-2-Amino-6-hydroxy-hexanoic acid (Chemstep) (5.54 mmol, 1.37 g) wasdissolved in 100 mL of MeOH and cooled at 0° C. SOCl₂ (13.8 mmol, 1 mL,2.5 eq) was added dropwise. The reaction mixture was stirred overnightat room temperature after which time the solvent was removed in vacuo.The crude solid was recrystallised from MeOH (3×100) to give the methylester 82 as an oil (5.52 mmol, 0.89 g, 99%).

(S)-6-Hydroxy-2-{(S)-2-[(S)-3-(4-hydroxy-phenyl)-2-methyl-propionylamino]-4-methyl-pentanoylamino}-hexanoicacid methyl ester (83)

To a suspension of(S)-2-[(S)-2-benzyloxycarbonylamino-3-(4-hydroxy-phenyl)-propionyl-amino]-4-methylpentanoic acid (Bachem) (5.44 mmol, 1.36 g, 1 eq) and methyl ester 82(5.44 mmol, 0.91 g) in DMF was added HATU (5.98 mmol, 1.41 g, 1.1 eq).DIPEA (0.12 mol, 1.62 g, 2.1 eq) was added and the reaction mixture wasstirred at room temperature overnight. EtOAc (100 mL) was added themixture was washed with 1M hydrochloric acid, saturated aqueous NaHCO₃and brine. The organic layer was dried over MgSO₄ and the solvent wasremoved in vacuo. The crude product was purified by recrystallisationfrom EtOAc to provide diol 83 as an oil (2.39 g, 76%).

Alternative Ring Closing Methods

The ring closing metathesis of dienes 70, 3, 22, 76 and 74 was examinedboth thermally and under microwave promoted conditions. The effect ofthe addition of the Lewis acid chloro-dicyclohexyl borane on thestereochemical outcome of the ring closing metathesis both thermally andunder microwave promoted conditions was also examined. The reactionswere performed under the following conditions:

A. Thermal reflux: to a solution of diene in anhydrous1,1,2-trichloroethane (0.01M) under an inert atmosphere was addedGrubb's second generation catalyst (10 mol %) and heated to reflux.After 1 hour a second portion of catalyst (10 mol %) was added andheated for another hour before the final portion (10 mol %) was added.The reaction mixture was heated at reflux for a further 16 hours,cooled, stirred overnight with activated charcoal, filtered andconcentrated in vacuo.

B. Microwave reflux: to a solution of diene in anhydrous1,1,2-trichloroethane (0.01M) under an inert atmosphere was addedGrubb's second generation catalyst (10 mol %) and heated for 20 minutesin a microwave (1200 W, 110-115° C.). Two more portions of catalyst(2×10 mol %) were added with 20 minutes heating between each addition.The reaction mixture was cooled, stirred overnight with activatedcharcoal, filtered and concentrated in vacuo.

C. Thermal reflux with Lewis acid: to a solution of diene in anhydrous1,1,2-trichloroethane (0.01M) under an inert atmosphere was addedGrubb's second generation catalyst (10 mol %) and chlorodicyclohexylborane (1M solution in hexane, 10 mol %) then heated to reflux. After 1hour a second portion of catalyst (10 mol %) was added and heated foranother hour before the final portion (10 mol %) was added. The reactionmixture was heated at reflux for a further 16 hours, cooled, stirredovernight with activated charcoal, filtered and concentrated in vacuo.

D. Microwave reflux with Lewis acid: to a solution of diene in anhydrous1,1,2-trichloroethane (0.01M) under an inert atmosphere was addedGrubb's second generation catalyst (10 mol %) and chlorodicyclohexylborane (1M solution in hexane, 10 mol %) then heated for 20 minutes in amicrowave (1200 W, 110-115° C.). Two more portions of catalyst (2×10 mol%) were added with 20 minutes heating between each addition. Thereaction mixture was cooled, stirred overnight with activated charcoal,filtered and concentrated in vacuo.

The stereochemical outcome of the ring closing metathesis (RCM) underthe different conditions and the yield of unsaturated macrocycles 71, 4,23, 77 and 74 is detailed in Table 1.

TABLE l^(a) Geometric Ring isomer Yield Diene RCM Product Size ratioE:Z^(a) (%)

A B C D

16  6:1 >19:1 >19:1 >19:1 49 58 50 48

A B C D

17  9:1  9:1  9:1  9:1 22 43 82 91

A B C D

18  1:1.7^(d)  1:1.7^(d)  1:1.7^(d)  1:1.9 51 58 74 100 

A B C D

19  6:1^(d)  4:1^(d)  5:1^(d) >19:1 22 24 30 33

A B C D

20  9:1  9:1 >19:1 >19:1 41 42 45 40 ^(a)Ratios measured using NMRintegrals. ^(b)Major geometric isomers assigned by NMR J couplingconstants. ^(c)Major geometric isomers unambiguously assigned by X-raycrystallography. ^(d)Three macrocyclic products were isolated from theRCM reaction of this diene. Two products were unambiguously assigned thestructure of the desired cis and trans olefins. The NMR data of thethird products clearly suggested double bond migration. ^(e)Majorgeometric isomers assigned by NOE experiments.

Enzyme Specificity

The inhibitors were assayed against several enzymes using aBODIPY-casein substrate in the fluorescence-based assay procedure of V.F. Thompson, S. Saldana, J. Cong and D. E. Goll, Anal. Biochem. 2000,279, 170. The results of these assays are presented in Table 2

TABLE 2 IC₅₀ calpain I calpain II Compound (μ calpain) (m calpain)cathepsin B pepsin α-chymotrypsin papain 8 1750 nM 700 nM 290 nM >50000nM >50000 nM >50000 nM 9 223 nM 30 nM 70 nM >25000 nM >50000 nM >50000nM 27 1340 nM 1100 nM 1100 nM >20000 nM >50000 nM >50000 nM 28 170 nM180 nM 770 nM >50000 nM >50000 nM >50000 nM 79 >50000 nM >50000nM >50000 nM >50000 nM >50000 nM >50000 nM 80 35000 nM 8900 nM 2400nM >50000 nM >50000 nM  12000 nM

Enzyme Inhibition

A number of the compounds were assayed against m-calpain using aBODIPY-casein substrate in the fluorescence-based assay procedure of V.F. Thompson, S. Saldana, J. Cong and D. E. Goll, Anal. Biochem. 2000,279, 170. The results of these assays are presented in Table 3.

The results of the enzyme inhibition assay for(7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamicacid benzyl ester (9) are graphically represented in FIG. 1 for whichy=30.924x+97.63 and R²=0.8554.

TABLE 3 Cmpd A R₂ R₃ R₂₀ R₅ R₆ IC₅₀ 9 C(═O)R₅ Leu CHO butyl benzyloxy —30 nM 28 C(═O)R₅ Leu CHO pentyl benzyloxy — 180 nM 50 C(═O)R₅ Val CHObutyl benzyloxy — 40 nM 52 C(═O)R₅ Leu CHO butyl pyrrolyl — 690 nM 17S(═O₂)R₆ Leu CHO butyl — 4F-Ph 45 nM 54 S(═O₂)R₆ Val CHO butyl — 4F-Ph280 nM 69 S(═O₂)R₆ Val CHO propyl — 4F-Ph 3710 nM 56 S(═O₂)R₆ Val CHObutyl — Me 950 nM 36 C(═O)R₅ Val CHO (CH₂)₄SCH₂ benzyloxy — 295 nM 58S(═O₂)R₆ Val CHO (CH₂)₄SCH₂ — 4F-Ph 2400 nM 8 C(═O)R₅ Leu CH₂OH butylbenzyloxy — 700 nM 27 C(═O)R₅ Leu CH₂OH pentyl benzyloxy — 1340 nM 16S(═O)R₆ Leu CH₂OH butyl — 4F-Ph 930 nM 79 C(═O)R₅ Leu CH₂N₃ butylbenzyloxy — >50000 nM 80 C(═O)R₅ Leu C(OH)CN butyl benzyloxy — 8900 nM81 C(═O)R₅ Leu CH₂NH —C₃H₂ butyl benzyloxy — >50000 nM

In Vitro Lens Culture Assay

The ability of compound 9 to prevent the formation of a calcium inducedcataract in adult ovine lens was assayed using the procedure of J.Sanderson, J. M. Marciantonio and G. A. Duncan, Invest. Opth. Vis. Sci.2000, 41, 2255.

Six pairs of lenses were tested. One lens from each pair waspreincubated with [1 μM] 9 in EMEM-culture media, for 3 h while theother was incubated at 35° C., 5% CO₂. Then 5 mM calcium chloride wasadded onto both the inhibitor treated lens and the other lens, and bothlenses were then incubated for 20 h. The lenses were photographed andthe images digitally analysed for opacity. The results of these studiesare presented in FIG. 2.

A typical pair of lenses was photographed when the assay had beencompleted. FIG. 3 shows the lens that was preincubated with compound 9prior to the addition of calcium chloride. FIG. 4 shows the other lens.

In Vivo Tests

An ointment (50 mg) comprising 1% of compound 9 was applied to one eyeof a lamb, three times in one day. No sign of irritation was observedand the lamb was then sacrificed.

A flock of 63 lambs genetically predisposed to cataracts were split intothree equal groups. An ointment (25 mg) comprising 1% of compound 9 wasapplied twice daily to the right eye of one of the groups of 21 lambsfor three months starting when they were three to four months old. Anointment (25 mg) comprising 1% of compound 8 was applied twice daily tothe right eye of another of the groups of 21 lambs for three monthsstarting when they were three to four months old. A placebo ointment (25mg) was applied twice daily to the right eye of the final group of 21lambs for three months starting when they were three to four months old.

The progression of cataracts was determined by a veterinaryophthalmologist with a slit-lamp microscope. The treated right eye didnot show significantly slower cataract progression than the left eye forany of the three groups. However, cataract progression in both eyes ofthe animals treated with the ointment containing compound 9 or with theointment containing compound 8 was significantly slower (p=0.066 andp=0.26, respectively) than the placebo-treated lambs. These results arepresented in FIG. 5.

Compound 9 was found to be capable of slowing cortical cataractprogression by 30% over a 12 week period in a flock of lambs geneticallypredisposed to cataracts with no signs of toxicity.

Formulations Ointment

An ointment, suitable for intraocular application, and having thefollowing composition (w/w) was prepared:

1% compound of Formula I25% cetyl stearyl alcohol35% wool fat39% paraffinum subl.

Cetyl stearyl alcohol was heated until it had melted. The compound ofFormula I was added and the oil stirred until the compound haddissolved. Wool fat and paraffinum subl. were added and the mixture washeated until all the components had melted. The mixture was allowed tocool with constant stirring until an ointment had formed.

Emulsion

An emulsion, suitable for intraocular application, and having thefollowing composition (w/w) was prepared according to the proceduredescribed below:

0.7% compound of Formula I20% cetyl stearyl alcohol25% wool fat25% paraffinum subl.1% sodium lauryl sulfate0.1% sodium benzoate28.3% water

The hydrophobic phase (cetyl stearyl alcohol, wool fat, paraffinumsubl.) and the hydrophilic phase (sodium lauryl sulfate, sodiumbenzoate, water) were separately heated to 50° C. The compound ofFormula I was added to the hydrophobic phase which was stirred until thecompound dissolved. The hydrophilic phase was then added to thehydrophobic phase, and the heating source removed. The mixture wasstirred until it reached rt. The resulting emulsion was then checked forthe absence of crystals by differential scanning calorimetry at themelting point of the compound of Formula I.

INDUSTRIAL APPLICATION

It will be appreciated from the discussion above that this inventionprovides novel compounds having cysteine protease inhibitory properties,more particularly calpain inhibitory properties. These compounds may beformulated into pharmaceutical compositions for use in any therapeuticapplication for which their inhibitory activity make them appropriate.Such therapeutic applications include the prevention or treatment ofcataracts.

Although the invention has been described by way of example and withreference to particular embodiments, it is to be understood thatmodifications and/or improvements may be made without departing from thescope of the invention as set out in the accompanying claims.

1.-64. (canceled)
 65. A compound of Formula I or a pharmaceuticallyacceptable salt, solvate, hydrate or prodrug derivative thereof:

wherein; A is —C(═O)R₅ or —S(═O)₂R₆; wherein R₅ is optionallysubstituted C₁-C₆ alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted heteroaryloxy, optionally substituted arylalkoxy oroptionally substituted heteroarylalkoxy; and R₆ is optionallysubstituted C₁-C₆ alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted heteroaryloxy, optionally substituted arylalkoxy oroptionally substituted heteroarylalkoxy; R₂ is a side chain of a naturalor non-natural alpha-amino acid; R₃ is —CH₂OH, —CH₂OR₇, —CH₂N₃,—CH₂NR₈R₉, —CH(OH)R₁₀, —CHO, —CH(OH)C(═O)NR₁₁R₁₂, —C(═O)C(═O)NR₁₁R₁₂, or—C(═O)R₁₃; wherein R₇ is C₁-C₆ alkyl, aryl or arylalkyl; R₈ is hydrogen,C₁-C₆ alkyl, aryl or arylalkyl; R₉ is hydrogen, C₁-C₆ alkyl, aryl orarylalkyl; R₁₀ is C₁-C₆ alkyl, alkoxy, thioalkoxy, aryl, arylalkyl,heteroaryl, heteroarylalkyl or cyano; R₁₁ and R₁₂ are each independentlyselected from hydrogen, C₁-C₆ alkyl, hydroxyalkyl, aryl, arylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl and heteroarylalkyl; or R₁₁and R₁₂ taken together with the nitrogen to which they are attached forma heterocyclyl or heteroaryl; and R₁₃ is C₁-C₆ alkyl, aryl, arylalkyl,heteroaryl or heteroarylalkyl; R₄ is —O—R₂₀— which is attached to the1,4-phenylene ring through the oxygen atom; wherein R₂₀ is optionallysubstituted straight chain —(C₃-C₆)-alkyl- or optionally substitutedstraight chain —(C₃-C₆)-alkenyl-; wherein any one methylene group withinthe straight chain —(C₃-C₆)-alkyl- or straight chain —(C₃-C₆)-alkenyl-,except the methylene group adjacent to the oxygen atom to which R₂₀ isattached, may be replaced by an oxygen, nitrogen or sulfur heteroatom ora —S(═O)— or —S(═O)₂— group; and wherein any two carbon atoms, or acarbon atom and a nitrogen heteroatom, if present, of the straight chain—(C₃-C₆)-alkyl- or straight chain —(C₃-C₆)-alkenyl- may be linked to oneanother through a chain of 1 to 4 atoms to form a fused ring selectedfrom optionally substituted cycloalkyl, optionally substitutedcycloalkenyl, optionally substituted heterocyclyl, optionallysubstituted aryl and optionally substituted heteroaryl; and R₆₀, R₆₁,R₆₂ and R₆₃ are each independently selected from hydrogen, halogen,—NH₂, —NO₂, —OH, C₁-C₆ alkyl and C₁-C₆ alkoxy.
 66. A compound as claimedin claim 65, wherein the compound has the following stereochemistry:


67. A compound as claimed in claim 65, wherein R₆₀, R₆₁, R₆₂ and R₆₃ areeach hydrogen.
 68. A compound as claimed in claim 65, wherein R₂ is aside chain of a natural alpha-amino acid.
 69. A compound as claimed inclaim 65, wherein R₂ is a side chain of L-leucine or L-valine.
 70. Acompound as claimed in claim 65, wherein A is —C(═O)R₅.
 71. A compoundas claimed in claim 70, wherein R₅ is optionally substituted arylalkoxy,optionally substituted aryl or heteroaryl.
 72. A compound as claimed inclaim 71, wherein R₅ is benzyloxy.
 73. A compound as claimed in claim71, wherein R₅ is 2-pyrrolyl.
 74. A compound as claimed in claim 65,wherein A is —S(═O)₂R₆.
 75. A compound as claimed in claim 74, whereinR₆ is optionally substituted C₁-C₆ alkyl or optionally substituted aryl.76. A compound as claimed in claim 75, wherein R₆ is methyl.
 77. Acompound as claimed in claim 75, wherein R₆ is 4-fluorophenyl.
 78. Acompound as claimed in claim 65, wherein R₃ is —CH₂OH or —CHO.
 79. Acompound as claimed in claim 65, wherein R₄ is —O-propyl-, —O-butyl-,—O-pentyl-, —O—(CH₂)₄OCH₂— or —O—(CH₂)₄SCH₂—.
 80. A compound selectedfrom the group consisting of:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.
 81. A compound selected from the group consistingof:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.
 82. The compound:

and the pharmaceutically acceptable salts, solvates, hydrates or prodrugderivatives thereof.
 83. A method for the treatment or prophylaxis of adisease or disorder resulting from excessive cysteine protease activityin a mammal comprising the step of administering a compound as claimedin claim 65 to the mammal.
 84. A method as claimed in claim 83, whereinthe cysteine protease is a calpain.
 85. A method as claimed in claim 84,wherein the disease or disorder is selected from the group consistingof: inflammatory and immunological diseases; cardiovascular andcerebrovascular diseases; disorders of the central or peripheral nervoussystem; osteoporosis; muscular dystrophies; cachexia; proliferativediseases; loss of hearing; ocular disorders; organ transplants;auto-immune and viral diseases; and cancer.
 86. A method for thetreatment or prophylaxis of cataracts in a mammal comprising the step ofadministering a compound as claimed in claim 65 to the mammal.
 87. Apharmaceutical composition comprising a compound as claimed in claim 65and a pharmaceutically acceptable carrier, diluent or excipient.